Macrocyclic inhibitors of flaviviridae viruses

ABSTRACT

Provided are compounds of Formula I: 
     
       
         
         
             
             
         
       
     
     and pharmaceutically acceptable salts and esters thereof. The compounds, compositions, and methods provided are useful for the treatment of virus infections, particularly hepatitis C infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 61/657,553, filed on Jun. 8, 2012, theentirety of which is incorporated herein by reference.

FIELD

The present application provides novel compounds inhibiting viruses,compositions containing such compounds, and therapeutic methodscomprising the administration of such compounds.

BACKGROUND

RNA viruses comprising the Flaviviridae family include at least threedistinguishable genera including pestiviruses, flaviviruses, andhepaciviruses (Calisher, et al., J. Gen. Virol., 1993, 70, 37-43). Whilepestiviruses cause many economically important animal diseases such asbovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV,hog cholera) and border disease of sheep (BDV), their importance inhuman disease is less well characterized (Moennig, V., et al., Adv. Vir.Res. 1992, 48, 53-98). Flaviviruses are responsible for important humandiseases such as dengue fever and yellow fever while hepaciviruses causehepatitis C virus infections in humans. Other important viral infectionscaused by the Flaviviridae family include West Nile virus (WNV) Japaneseencephalitis virus (JEV), tick-borne encephalitis virus, Junjin virus,Murray Valley encephalitis, St Louis enchaplitis, Omsk hemorrhagic fevervirus and Zika virus.

The hepatitis C virus (HCV) is the leading cause of chronic liverdisease worldwide (Boyer, N. et al. J Hepatol. 32:98-112, 2000) so asignificant focus of current antiviral research is directed toward thedevelopment of improved methods of treatment of chronic HCV infectionsin humans (Di Besceglie, A. M. and Bacon, B. R., Scientific American,October: 80-85, (1999); Gordon, C. P., et al., J. Med. Chem. 2005, 48,1-20; Maradpour, D., et al., Nat. Rev. Micro. 2007, 5(6), 453-463). Anumber of HCV treatments are reviewed by Dymock et al. in AntiviralChemistry & Chemotherapy, 11:2; 79-95 (2000). Virologic cures ofpatients with chronic HCV infection are difficult to achieve because ofthe prodigious amount of daily virus production in chronically infectedpatients and the high spontaneous mutability of HCV virus (Neumann, etal., Science 1998, 282, 103-7; Fukimoto, et al., Hepatology, 1996, 24,1351-4; Domingo, et al., Gene 1985, 40, 1-8; Martell, et al., J. Virol.1992, 66, 3225-9.

Currently, there are primarily two antiviral compounds, ribavirin, anucleoside analog, and interferon-alpha (a) (IFN), that are used for thetreatment of chronic HCV infections in humans. Ribavirin alone is noteffective in reducing viral RNA levels, has significant toxicity, and isknown to induce anemia. The combination of IFN and ribavirin has beenreported to be effective in the management of chronic hepatitis C(Scott, L. J., et al. Drugs 2002, 62, 507-556) but less than half thepatients given this treatment show a persistent benefit. Therefore,there is a need to develop more effective anti-HCV therapies.

The macrocycle sanglifehrin and derivatives are immunomodulatory andbind peptidyl-prolyl cis/trans isomerase (PPlase) cyclophilins in aunique manner (WO 97/02285; WO 98/07743; J. Am. Chem. Soc 2003, 125,3849-3859; J. Org. Chem. 2000, 65, 9255-9260; Angew. Chem. Int. Ed.1999, 38, 2443-2446). The cyclophilins are peptidyl-prolyl cis/transisomerases (PPlase) that regulate protein folding in vivo and inhibithepatitis C virus (Lin et al., WO2006/138507). However, none of thesanglifehrins or their derivatives has become available for humananti-viral therapy. Therefore, there is a continuing need to developmacrocyclic sanglifehrins with anti-Flaviviridae virus activity andparticularly anti-HCV activity.

SUMMARY

In one embodiment, there is provided a compound represented by FormulaI:

or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture ofstereoisomers, tautomer, ester or prodrug thereof, wherein:

A¹ is (C₂-C₅)alkylene, (C₂-C₅)alkenylene, (C₂-C₅)alkynylene,—O—(C₂-C₄)alkylene, —O—(C₂-C₄)alkenylene, arylene, aryl(C₁-C₂)alkylene,heterocycloalkylene or heterocycloalkyl(C₁-C₂)alkylene, wherein a spacarbon atom of A¹ is optionally substituted with one or more(C₁-C₄)alkyl;

A² is arylene or heteroarylene, wherein A² is optionally substitutedwith halo;

X¹ is —O—, —NH— or —N((C₁-C₄)alkyl)-;

R^(1a) and R^(1b) are independently H, (C₁-C₄)alkyl, (C₂-C₄)alkenyl or(C₂-C₄)alkynyl;

R² is H, (C₁-C₄)alkyl, (C₂-C₄)alkenyl or (C₂-C₄)alkynyl;

R^(3a) and R^(3b) are independently H or (C₁-C₈)alkyl;

R^(4a) and R^(4b) are independently H, —OH, (C₁-C₄)alkoxy,halo(C₁-C₄)alkoxy or (C₁-C₈)alkyl;

R⁵ is H, (C₁-C₄)alkyl, (C₂-C₄)alkenyl or (C₂-C₄)alkynyl, or

R⁵ forms a cyclic moiety along with —N((C₁-C₄)alkyl)- of X¹ or aryleneof A²; and

R⁶ is H or (C₁-C₄)alkyl.

In another embodiment, there is provided a pharmaceutical compositioncomprising a compound of Formula I r a pharmaceutically acceptable salt,isotope, stereoisomer, mixture of stereoisomers, tautomer, ester orprodrug thereof and one or more pharmaceutically acceptable carriers orexcipients. In one aspect of the embodiment, the pharmaceuticalcomposition further comprises one or more additional therapeutic agents.

In yet another embodiment, a method for treating Flaviviridae viralinfection is provided comprising administering a therapeuticallyeffective amount of a compound of Formula I r a pharmaceuticallyacceptable salt, isotope, stereoisomer, mixture of stereoisomers,tautomer, ester or prodrug thereof to a mammal in need thereof. In oneaspect of the embodiment, the treatment results in the reduction of thein viral load or clearance of viral RNA in a patient.

In yet another embodiment, a method for treating Coronaviridae viralinfection is provided comprising administering a therapeuticallyeffective amount of a compound of Formula I r a pharmaceuticallyacceptable salt, isotope, stereoisomer, mixture of stereoisomers,tautomer, ester or prodrug thereof to a mammal in need thereof. In oneaspect of the embodiment, the treatment results in the reduction of thein viral load or clearance of viral RNA in a patient.

DETAILED DESCRIPTION Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

“Alkanoyl” is RC(O)—; “alkanoyloxy” is RC(O)O—; and “alkanoylamino” isRC(O)NR′—; where R is an alkyl group as defined herein, and R′ ishydrogen or alkyl.

“Alkenyl” refers to a straight or branched hydrocarbyl group with atleast one site of unsaturation, i.e., a carbon-carbon, sp² double bond.In some embodiments, alkenyl is a C₂-C₂₀ alkenyl group, a C₂-C₁₀ alkenylgroup or a C₂-C₆ alkenyl group. Examples of alkenyl group include, butare not limited to, vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl(—C₅H₇), and 5-hexenyl (—CH₂CH₂CH₂CH₂CH═CH₂).

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical having two monovalent radical centers derivedby the removal of two hydrogen atoms from the same or two differentcarbon atoms of a parent alkene. For example, and alkenylene group canhave 2 to 20 carbon atoms, 2 to 10 carbon atoms, or 2 to 6 carbon atoms.Typical alkenylene radicals include, but are not limited to,1,2-ethenylene (—CH═CH—).

“Alkoxy” is RO— where R is alkyl, as defined herein. Non-limitingexamples of alkoxy groups include methoxy, ethoxy and propoxy.

“Alkyl” refers to a straight or branched chain hydrocarbyl group. In anembodiment, alkyl has from 1 to 20 carbon atoms (i.e., C₁-C₂₀ alkyl). Insome embodiments, alkyl is a C₁-C₁₀ alkyl group or a C₁-C₆ alkyl group.Examples of alkyl groups include, but are not limited to, methyl, ethyl,propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl,octyl, nonyl and decyl.

“Alkylene” refers to a saturated, branched or straight chain radical orcyclic hydrocarbon radical having two monovalent radical centers derivedby the removal of two hydrogen atoms from the same or two differentcarbon atoms of a parent alkane. For example, an alkylene group can have1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms.Examples of alkylene radicals include, but are not limited to, methylene(—CH₂—), ethylene (—CH₂CH₂—), propylene (—CH₂CH₂CH₂—) and butylene(—CH₂CH₂CH₂CH₂—).

“Alkynyl” refers to a hydrocarbon containing normal, secondary ortertiary carbon atoms with at least one site of unsaturation, i.e., acarbon-carbon, sp triple bond. For example, an alkynyl group can have 2to 20 carbon atoms (i.e., C₂-C₂₀ alkynyl), 2 to 12 carbon atoms (i.e.,C₂-C₁₂ alkyne,) or 2 to 6 carbon atoms (i.e., C₂-C₆ alkynyl). Examplesof alkynyl groups include, but are not limited to, acetylenic (—CH) andpropargyl (—CH₂C≡CH).

“Alkylamino” refers to an amino group substituted with one or more alkylgroups. “Mono(alkyl)amino” or “(alkyl)amino” is RNH—, and“di(alkyl)amino” or “(alkyl)₂amino” is R₂N—, where each of the R groupsis alkyl as defined herein and are the same or different. Examples ofalkylamino groups include, but are not limited to, methylamino,ethylamino, propylamino, butylamino, dimethylamino, diethylamino andmethylethylamno.

“Amino” refers to —NH₂.

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical having two monovalent radical centers derivedby the removal of two hydrogen atoms from the same or two differentcarbon atoms of a parent alkyne. For example, an alkynylene group canhave 2 to 20 carbon atoms, 2 to 10 carbon atoms, or 2 to 6 carbon atoms.Typical alkynylene radicals include, but are not limited to, acetylene(—C≡C—), propargylene (—CH₂C≡C—), and 4-pentynylene (—CH₂CH₂CH₂C≡C—).

“Aryl” refers to any monocyclic or bicyclic carbon ring of up to 7 atomsin each ring, wherein at least one ring is aromatic, or an aromatic ringsystem of 5 to 14 carbons atoms which includes a carbocyclic aromaticgroup fused with a 5- or 6-membered cycloalkyl group. Examples of arylgroups include, but are not limited to, phenyl, naphthyl,tetrahydronaphthyl and indanyl.

“Arylalkyl” refers to an alkyl as defined herein substituted with anaryl radical.

“Arylene” refers to an aryl as defined above having two monovalentradical centers derived by the removal of two hydrogen atoms from twodifferent carbon atoms of a parent aryl. Typical arylene radicalsinclude, but are not limited to, phenylene, e.g.,

and naphthylene, e.g.,

“Arylalkylene” refers to an arylalkyl as defined above having twomonovalent radical centers derived by the removal of one hydrogen atomfrom the aryl radical and the other hydrogen removed from the alkylradical of the group.

“Cycloalkyl” refers to a hydrocarbyl group containing at least onesaturated or partially unsaturated ring structure, and attached via aring carbon. Cycloalkyl groups include hydrocarbon mono-, bi-, andpoly-cyclic rings, whether fused, bridged, or spiro. In variousembodiments, it refers to a saturated or a partially unsaturated C₃-C₁₂cyclic moiety, examples of which include cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl andcyclooctyl.

“Cycloalkylalkyl” refers to an alkyl moiety substituted with acycloalkyl group. Examples of cycloalkylalkyl groups includecyclopropylmethyl, cyclobutylmethyl, cyclopentylethyl andcyclohexylmethyl.

“Cycloalkylene” refers to a cycloalkyl, as defined herein, having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent cycloalkyl.Examples of cycloalkylene include, but are not limited to,cyclopropylene, cyclobutylene, cyclopentylene and cyclohexylene.

“Dialkylaminoalkyl” refers to an alkyl moiety substituted with adialkylamino group, wherein dialkylamino is as defined herein.

“Ester” means any ester of a compound in which any of the —COOHfunctions of the molecule is replaced by a —C(O)OR function, or in whichany of the —OH functions of the molecule are replaced with a —OC(O)Rfunction, in which the R moiety of the ester is any carbon-containinggroup which forms a stable ester moiety, including but not limited toalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl,heterocyclyl, heterocyclylalkyl and substituted derivatives thereof.

“Halo” refers to chloro (—Cl), bromo (—Br), fluoro (—F) or iodo (—I).

“Haloalkoxy” refers to alkoxy, as defined herein, substituted with oneor more halo radicals.

“Haloalkoxyalkyl” refers to an alkyl moiety substituted with ahaloalkoxy group, as defined herein.

“Haloalkyl” refers to an alkyl group, in which one or more hydrogenatoms of the alkyl group is replaced with a halogen atom. Examples ofhaloalkyl groups include, but are not limited to, —CF₃, —CHF₂, —CFH₂ and—CH₂CF₃.

“Heterocycloalkyl” refers to a saturated or partially unsaturatedmonocyclic, bicyclic or tricyclic group of 2 to 14 ring-carbon atomsand, in addition to ring-carbon atoms, 1 to 4 heteroatoms selected fromP, N, O and S. The heterocyclic group can be attached through a carbonatom or through a heteroatom, and when substituted, the substituent canbe bonded to a carbon atom or a heteroatom. Examples of heterocyclylinclude azetidinyl, benzoimidazolyl, benzofuranyl, benzofurazanyl,benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl,indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl,pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl,tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl,triazolyl, azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl,piperidinyl, pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzoimidazolyl, di hydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl andN-oxides thereof.

“Heterocycloalkylene” refers to a heterocycloalkyl, as defined above,having two monovalent radical centers derived by the removal of twohydrogen atoms from the same or two different carbon atoms of a parentheterocycloalkyl group.

“Heterocycloalkylalkylene” refers to an heterocycloalkyl as definedabove having two monovalent radical centers derived by the removal ofone hydrogen atom from the heterocycloalkyl radical and the otherhydrogen removed from the alkyl radical of the group.

“Heteroaryl” refers to a monocyclic, bicyclic or tricyclic ring havingup to 7 atoms in each ring, wherein at least one ring is aromatic andcontains from 1 to 4 heteroatoms in the ring selected from the groupconsisting of N, O and S. Non-limiting examples of heteroaryl includepyridyl, thienyl, furanyl, pyrimidyl, imidazolyl, pyranyl, pyrazolyl,thiazolyl, thiadiazolyl, isothiazolyl, oxazolyl, isoxazoyl, pyrrolyl,pyridazinyl, pyrazinyl, quinolinyl, isoquinolinyl, benzofuranyl,dibenzofuranyl, dibenzothiophenyl, benzothienyl, indolyl,benzothiazolyl, benzooxazolyl, benzimidazolyl, isoindolyl,benzotriazolyl, purinyl, thianaphthenyl and pyrazinyl. Attachment ofheteroaryl can occur via an aromatic ring, or, if heteroaryl is bicyclicor tricyclic and one of the rings is not aromatic or contains noheteroatoms, through a non-aromatic ring or a ring containing noheteroatoms. “Heteroaryl” is also understood to include the N-oxidederivative of any nitrogen containing heteroaryl.

“Heteroarylalkyl” refers to an alkyl group, as defined herein, in whicha hydrogen atom has been replaced with a heteroaryl group.

“Heteroarylene” refers to a heteroaryl, as defined above, having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent heteroarylgroup. Non-limiting examples of heteroarylene groups are:

“Hydroxyalkoxy” refers to an alkoxy, as defined herein, substituted witha hydroxyl group (—OH). An example of hydroxyalkoxy is hydroxyethoxy.

“Hydroxyalkyl” refers to an alkyl group substituted with at least onehydroxy group. Examples of hydroxyalkyl groups include, but are notlimited to, hydroxymethyl, hydroxyethyl, hydroxypropyl and hydroxybutyl.

“Prodrug” refers to any compound that when administered to a biologicalsystem generates the drug substance, i.e., active ingredient, as aresult of spontaneous chemical reaction(s), enzyme catalyzed chemicalreaction(s), photolysis, and/or metabolic chemical reaction(s). Aprodrug is thus a covalently modified analog or latent form of atherapeutically active compound. Non-limiting examples of prodrugsinclude ester moieties, quaternary ammonium moieties, glycol moieties,and the like.

The term “optionally substituted” refers to a moiety wherein allsubstituents are hydrogen or wherein one or more of the hydrogens of themoiety are replaced by non-hydrogen substituents. Multiple substitutionson the same atom are also permitted where chemically feasible (e.g., adioxo substitution to provide —S(O)₂—, geminal substituents, spirocycloalkyl or heterocycloalkyl rings, etc.). In some embodiments, “oneor more” substituents is from one to three substituents.

Embraced herein, where applicable, are permissible isomers such astautomers, racemates, enantiomers, diastereomers, atropisomers,configurational isomers of double bonds (E- and/or Z-), cis- andtrans-configurations in ring substitution patterns, and isotopicvariants.

“Pharmaceutically acceptable” means suitable for use in pharmaceuticalpreparations, generally considered as safe for such use, officiallyapproved by a regulatory agency of a national or state government forsuch use, or being listed in the U. S. Pharmacopoeia or other generallyrecognized pharmacopoeia for use in animals, and more particularly inhumans.

“Pharmaceutically acceptable carrier” refers to a diluent, adjuvant,excipient, or carrier, or other ingredient which is pharmaceuticallyacceptable and with which a compound of the invention is administered.

“Pharmaceutically acceptable salt” refers to a salt which may enhancedesired pharmacological activity. Examples ofpharmaceutically-acceptable salts include acid addition salts formedwith inorganic or organic acids, metal salts and amine salts. Examplesof acid addition salts formed with inorganic acids include salts withhydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid andphosphoric acid. Examples of acid addition salts formed with organicacids such as acetic acid, propionic acid, hexanoic acid, heptanoicacid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lacticacid, malonic acid, succinic acid, malic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid,o-(4-hydroxy-benzoyl)-benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethane-sulfonic acid, benzenesulfonic acid,p-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,p-toluenesulfonic acid, camphorsulfonic acid,4-methyl-bicyclo[2.2.2]oct-2-ene1-carboxylic acid, gluco-heptonic acid,4,4′-methylenebis(3-hydroxy-2-naphthoic) acid, 3-phenylpropionic acid,trimethyl-acetic acid, tertiary butylacetic acid, lauryl sulfuric acid,gluconic acid, glutamic acid, hydroxy-naphthoic acids, salicylic acid,stearic acid and muconic acid. Examples of metal salts include saltswith sodium, potassium, calcium, magnesium, aluminum, iron, and zincions. Examples of amine salts include salts with ammonia and organicnitrogenous bases strong enough to form salts with carboxylic acids.

A compound of a given formula (e.g. the compound of Formula I, whichalso includes Formula I-a, I-b, I-c, I-d, I-e or II) is intended toencompass the compounds of the disclosure, and the pharmaceuticallyacceptable salts, stereoisomers, mixture of stereoisomers or tautomersof such compounds. Additionally, the compounds of the disclosure maypossess one or more asymmetric centers, and can be produced as a racemicmixture or as individual enantiomers or diastereoisomers. The number ofstereoisomers present in any given compound of a given formula dependsupon the number of asymmetric centers present (there are 2″stereoisomers possible where n is the number of asymmetric centers). Theindividual stereoisomers may be obtained by resolving a racemic ornon-racemic mixture of an intermediate at some appropriate stage of thesynthesis or by resolution of the compound by conventional means. Theindividual stereoisomers (including individual enantiomers anddiastereoisomers) as well as racemic and non-racemic mixtures ofstereoisomers are encompassed within the scope of the presentdisclosure, all of which are intended to be depicted by the structuresof this specification unless otherwise specifically indicated.

“Isomers” are different compounds that have the same molecular formula.Isomers include stereoisomers, enantiomers and diastereomers.

“Stereoisomers” are isomers that differ only in the way the atoms arearranged in space. Stereoisomers include enantiomers and diastereomers.

“Enantiomers” are a pair of stereoisomers that are non-superimposablemirror images of each other. A 1:1 mixture of a pair of enantiomers is a“racemic” mixture. The term “(±)” is used to designate a racemic mixturewhere appropriate.

“Diastereoisomers” are stereoisomers that have at least two asymmetricatoms, but which are not mirror-images of each other.

The absolute stereochemistry is specified according to the Cahn IngoldPrelog R S system. When the compound is a pure enantiomer thestereochemistry at each chiral carbon may be specified by either R or S.Resolved compounds whose absolute configuration is unknown aredesignated (+) or (−) depending on the direction (dextro- orlaevorotary) that they rotate the plane of polarized light at thewavelength of the sodium D line.

Some of the compounds exist as tautomeric isomers or “tautomers”.Tautomeric isomers are in equilibrium with one another. For example,amide containing compounds may exist in equilibrium with imidic acidtautomers. Regardless of which tautomer is shown, and regardless of thenature of the equilibrium among tautomers, the compounds are understoodby one of ordinary skill in the art to comprise both amide and imidicacid tautomers. Thus, the amide containing compounds are understood toinclude their imidic acid tautomers. Likewise, the imidic acidcontaining compounds are understood to include their amide tautomers.

Any formula or structure provided herein, including Formula I, I-a, I-b,I-c, I-d, I-e or II, is also intended to represent unlabeled forms aswell as isotopically labeled forms of the compounds. An “isotope” mayhave structures depicted by the formulas given herein except that one ormore atoms are replaced by an atom having a selected atomic mass or massnumber. Examples of isotopes that can be incorporated into compounds ofthe disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine and chlorine, such as, but not limited to ²H(deuterium, D), ³H (tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F, ³¹P, ³²P, ³⁵S,³⁶Cl and ¹²⁵I. Various isotopically labeled compounds of the presentdisclosure, for example those into which radioactive isotopes such as³H, ¹³C and ¹⁴C are incorporated. Such isotopically labelled compoundsmay be useful in metabolic studies, reaction kinetic studies, detectionor imaging techniques, such as positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT) including drug orsubstrate tissue distribution assays or in radioactive treatment ofpatients.

The disclosure also included compounds of Formula I, I-a, I-b, I-c, I-d,I-e or II in which from 1 to n hydrogens attached to a carbon atomis/are replaced by deuterium, in which n is the number of hydrogens inthe molecule. Such compounds exhibit increased resistance to metabolismand are thus useful for increasing the half life of any compound ofFormul Formula I, I-a, I-b, I-c, I-d, I-e or II when administered to amammal. See, for example, Foster, “Deuterium Isotope Effects in Studiesof Drug Metabolism”, Trends Pharmacol. Sci. 5(12):524-527 (1984). Suchcompounds are synthesized by means well known in the art, for example byemploying starting materials in which one or more hydrogens have beenreplaced by deuterium.

Deuterium labelled or substituted therapeutic compounds of thedisclosure may have improved DMPK (drug metabolism and pharmacokinetics)properties, relating to distribution, metabolism and excretion (ADME).Substitution with heavier isotopes such as deuterium may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life, reduced dosage requirements and/oran improvement in therapeutic index. An ¹⁸F labeled compound may beuseful for PET or SPECT studies. Isotopically labeled compounds of thisdisclosure and prodrugs thereof can generally be prepared by carryingout the procedures disclosed in the schemes or in the examples andpreparations described below by substituting a readily availableisotopically labeled reagent for a non-isotopically labeled reagent. Itis understood that deuterium in this context is regarded as asubstituent in the compound of Formula I, I-a, I-b, I-c, I-d, I-e or II.

The concentration of such a heavier isotope, specifically deuterium, maybe defined by an isotopic enrichment factor. In the compounds of thisdisclosure any atom not specifically designated as a particular isotopeis meant to represent any stable isotope of that atom. Unless otherwisestated, when a position is designated specifically as “H” or “hydrogen”,the position is understood to have hydrogen at its natural abundanceisotopic composition. Accordingly, in the compounds of this disclosureany atom specifically designated as a deuterium (D) is meant torepresent deuterium.

“Therapeutically-effective amount” refers to an amount of a compoundthat, when administered to a subject for treating a disease, issufficient to effect treatment for the disease.

“Therapeutically effective amount” can vary depending on the compound,the disease and its severity, the age, the weight, etc. of the subjectto be treated.

The term “treating”, and grammatical equivalents thereof, when used inthe context of treating a disease, means slowing or stopping theprogression of a disease, or ameliorating at least one symptom of adisease, more preferably ameliorating more than one symptom of adisease. For example, treatment of a hepatitis C virus infection caninclude reducing the HCV viral load in an HCV infected human being,and/or reducing the severity of jaundice present in an HCV infectedhuman being.

Compounds

The present application provides a compound represented by Formula I:

or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture ofstereoisomers, tautomer, ester or prodrug thereof, wherein:

A¹ is (C₂-C₅)alkylene, (C₂-C₅)alkenylene, (C₂-C₅)alkynylene,—O—(C₂-C₄)alkylene, —O—(C₂-C₄)alkenylene, arylene, aryl(C₁-C₂)alkylene,heterocycloalkylene or heterocycloalkyl(C₁-C₂)alkylene, wherein a spacarbon atom of A¹ is optionally substituted with one or more(C₁-C₄)alkyl;

A² is arylene or heteroarylene, wherein A² is optionally substitutedwith halo;

X¹ is —O—, —NH— or —N((C₁-C₄)alkyl)-;

R^(1a) and R^(1b) are independently H, (C₁-C₄)alkyl, (C₂-C₄)alkenyl or(C₂-C₄)alkynyl;

R² is H, (C₁-C₄)alkyl, (C₂-C₄)alkenyl or (C₂-C₄)alkynyl;

R^(3a) and R^(3b) are independently H or (C₁-C₈)alkyl;

R^(4a) and R^(4b) are independently H, —OH, (C₁-C₄)alkoxy,halo(C₁-C₄)alkoxy or (C₁-C₈)alkyl;

R⁵ is H, (C₁-C₄)alkyl, (C₂-C₄)alkenyl or (C₂-C₄)alkynyl, or

R⁵ forms a cyclic moiety along with —N((C₁-C₄)alkyl)- of X¹ or aryleneof A²; and

R⁶ is H or (C₁-C₄)alkyl.

In certain embodiments, provided is a compound of Formula I:

or a pharmaceutically acceptable salt, ester or prodrug thereof,wherein:

A¹ is (C₁-C₅)alkylene, (C₂-C₅)alkenylene, (C₂-C₅)alkynylene,—O—(C₂-C₄)alkylene, —O—(C₂-C₄)alkenylene, arylene, aryl(C₁-C₂)alkylene,heterocycloalkylene or heterocycloalkyl(C₁-C₂)alkylene, wherein a spacarbon atom of A¹ is optionally substituted with one or more(C₁-C₄)alkyl;

A² is arylene or heteroarylene, wherein A² is optionally substitutedwith halo;

X¹ is —O—, —NH— or —N((C₁-C₄)alkyl)-;

R^(1a) and R^(1b) are independently H, (C₁-C₄)alkyl, (C₂-C₄)alkenyl or(C₂-C₄)alkynyl;

R² is H, (C₁-C₄)alkyl, (C₂-C₄)alkenyl or (C₂-C₄)alkynyl;

R^(3a) and R^(3b) are independently H or (C₁-C₈)alkyl;

R^(4a) and R^(4b) are independently H, —OH, (C₁-C₄)alkoxy,halo(C₁-C₄)alkoxy or (C₁-C₈)alkyl;

R⁵ is H, (C₁-C₄)alkyl, (C₂-C₄)alkenyl or (C₂-C₄)alkynyl, or

R⁵ forms a cyclic moiety along with —N((C₁-C₄)alkyl)- of X¹ or aryleneof A²; and

R⁶ is H or (C₁-C₄)alkyl.

In one aspect of the embodiment, A¹ is ethenylene, propenylene,butenylene, ethylene, propylene, butylene, oxypropylene, oxypropenylene,pyrazolylene, phenylene or pyrimidinylene.

In another aspect of the embodiment, A² is heteroarylene such asisoquinolinylene, phenylene or halophenylene. In one aspect, A² is

In another aspect of the embodiment, X¹ is —O— or —NH—; R^(1a) is H; Ribis methyl; R² is iso-propyl; R⁵ is methyl and R⁶ is H or methyl.

In another aspect of the embodiment, R^(3a) is H or methyl; R^(3b) is H;R^(4a) is H, —OH, methoxy, trifluoroethoxy; and R^(4b) is H.

In another aspect of the embodiment, A² is heteroarylene; A¹ is(C₂-C₅)alkylene, (C₂-C₅)alkenylene or (C₂-C₅)alkynylene, wherein A¹ isoptionally substituted with one or more (C₁-C₄)alkyl; R^(3a) is H or(C₁-C₈)alkyl; and R^(4a) is H, —OH or (C₁-C₄)alkoxy. In another aspectof the embodiment, A² is heteroarylene; A¹ is (C₁-C₅)alkylene,(C₂-C₅)alkenylene or (C₂-C₅)alkynylene, wherein A¹ is optionallysubstituted with one or more (C₁-C₄)alkyl; R^(3a) is H or (C₁-C₈)alkyl;and R^(4a) is H, —OH or (C₁-C₄)alkoxy. Non-limiting examples of suchcompounds include the following compounds and pharmaceuticallyacceptable salts thereof.

Compound No. X¹ A¹ R^(3a) R^(4a) a-1 —NH— —CH₂CH₂— methyl methoxy a-2—NH— —CH═CH— methyl methoxy a-3 —O— —CH₂CH₂— methyl methoxy a-4 —O——CH═CH— methyl methoxy a-5 —NH— —CH₂CH₂— methyl —H a-6 —NH— —CH═CH—methyl —H a-7 —O— —CH₂CH₂— methyl —H a-8 —O— —CH═CH— methyl —H a-9 —NH——CH═CHCH₂— —H —H a-10 —NH— —CH₂CH₂CH₂— —H —H a-11 —O— —CH═CHCH₂— —H —Ha-12 —O— —CH₂CH₂CH₂— —H —H a-13 —NH— —CH═CHCH₂CH₂— —H —H a-14 —NH——CH₂CH₂CH₂CH₂— —H —H a-15 —O— —CH═CHCH₂CH₂— —H —H a-16 —O——CH₂CH₂CH₂CH₂— —H —Hwherein the left bond of A¹ linker is attached to A².

In another aspect of the embodiment, A² is arylene; and A¹ is(C₂-C₅)alkylene, (C₂-C₅)alkenylene, (C₂-C₅)alkynylene,—O—(C₂-C₅)alkylene or —O—(C₂-C₄)alkenylene, wherein A¹ is optionallysubstituted with one or more (C₁-C₄)alkyl; R^(3a) is H or (C₁-C₄)alkyl;and R^(4a) is H, —OH, (C₁-C₄)alkoxy or halo(C₁-C₄)alkoxy. In anotheraspect of the embodiment, A² is arylene; and A¹ is (C₁-C₅)alkylene,(C₂-C₅)alkenylene, (C₂-C₅)alkynylene, —O—(C₂-C₅)alkylene or—O—(C₂-C₄)alkenylene, wherein A¹ is optionally substituted with one ormore (C₁-C₄)alkyl; R^(3a) is H or (C₁-C₄)alkyl; and R^(4a) is H, —OH,(C₁-C₄)alkoxy or halo(C₁-C₄)alkoxy. Non-limiting examples of suchcompounds include the following compounds and pharmaceuticallyacceptable salts thereof.

No. X¹ A¹ R^(1b) R^(3a) R^(4a) R⁵ b-1 —NH— —CH═CHCH₂CH₂— —H —H —OHmethyl b-2 —NH— —CH₂CH₂CH₂CH₂— —H —H —OH methyl b-3 —O— —CH═CHCH₂CH₂— —H—H —OH methyl b-4 —O— —CH₂CH₂CH₂CH₂— —H —H —OH methyl b-5 —NH——CH═CHCH₂CH₂— —H —H —OCH₂CF₃ methyl b-6 —NH— —CH₂CH₂CH₂CH₂— —H —H—OCH₂CF₃ methyl b-7 —O— —CH═CHCH₂CH₂— —H —H —OCH₂CF₃ methyl b-8 —O——CH₂CH₂CH₂CH₂— —H —H —OCH₂CF₃ methyl b-9 —NH— —CH═CHCH₂C(CH₃)₂— —H —H —Hmethyl b-10 —O— —CH═CHCH₂C(CH₃)₂— —H —H —H methyl b-11 —NH— —OCH₂CH═CH——H methyl methoxy isopropyl b-12 —O— —OCH₂CH═CH— —H methyl methoxyisopropyl b-13 —NH— —OCH₂CH═CH— —H methyl methoxy methyl b-14 —O——OCH₂CH═CH— —H methyl methoxy methyl b-15 —NH— —CH═CHCH₂CH₂— methyl —H—OH methyl b-16 —NH— —CH₂CH₂CH₂CH₂— methyl —H —OH methyl

wherein the left bond of A¹ linker is attached to A².

Also included are the following compounds and pharmaceuticallyacceptable salts thereof.

No. X¹ A¹ R^(3a) R^(4a) c-1 —NH— —CH═CHCH₂CH₂— methyl methoxy c-2 —NH——CH₂CH₂CH₂CH₂— methyl methoxy c-3 —O— —CH═CHCH₂CH₂— methyl methoxy c-4—O— —CH₂CH₂CH₂CH₂— methyl methoxy c-5 —NH— —CH═CHCH₂CH₂— —H —OH c-6 —NH——CH₂CH₂CH₂CH₂— —H —OH c-7 —O— —CH═CHCH₂CH₂— —H —OH c-8 —O——CH₂CH₂CH₂CH₂— —H —OH

wherein the left bond of A¹ linker is attached to A².

In another aspect of the embodiment, A² is phenylene; and A¹ ispyrazolylene, phenylene or pyrimidinylene. Non-limiting examples of suchcompounds include the following compounds and pharmaceuticallyacceptable salts thereof.

Compound No. X¹ A¹ R^(3a) R^(4a) d-1 —O—

—H —H d-2 —O—

—H —H d-3 —O—

—H —H d-4 —O—

—H —H d-5 —NH—

—H —H d-6 —NH—

—H —H d-7 —NH—

—H —H d-8 —NH—

—H —H

wherein the left bond of A¹ linker is attached to A².

In another aspect of the embodiment, A² is halophenylene; and A¹ is—O—(C₂-C₅)alkylene or —O—(C₂-C₄)alkenylene. Non-limiting examples ofsuch compounds include the following compounds and pharmaceuticallyacceptable salts thereof.

Compound No. X¹ A¹ R^(3a) R^(4a) e-1 —NH— —OCH₂CH═CH— methyl —H e-2 —O——OCH₂CH═CH— methyl —H e-3 —NH— —OCH₂CH₂CH₂— methyl —H e-4 —O——OCH₂CH₂CH₂— methyl —H e-5 —NH— —OCH₂CH═CH— methyl methoxy e-6 —O——OCH₂CH═CH— methyl methoxy e-7 —NH— —OCH₂CH₂CH₂— methyl methoxy e-8 —O——OCH₂CH₂CH₂— methyl methoxy

wherein the left bond of A¹ linker is attached to A².

In one aspect of the embodiment, A¹ is (C₂-C₅)alkylene or(C₂-C₅)alkenylene; R⁵ is methyl, or R⁵ form

along with arylene of A², or R⁵ form

along with —N((C₁-C₄)alkyl)- of X¹; and R⁶ is H or methyl. In anotheraspect of the embodiment, A¹ is (C₁-C₅)alkylene or (C₂-C₅)alkenylene; R⁵is methyl, or R⁵ form

along with arylene of A², or R⁵ form

along with —N((C₁-C₄)alkyl)- of X¹; and R⁶ is H or methyl. Non-limitingexamples of such compounds include the following compounds andpharmaceutically acceptable salts thereof:

In another embodiment, there is provided a compound of Formula II:

or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture ofstereoisomers, tautomer, ester or prodrug thereof, wherein:

A¹ is ethenylene,

wherein the left bond of A¹ linker is attached to A²;

A² is

X¹ is —O— or —NH—;

R^(3a) is H or (C₁-C₄)alkyl;

R^(4a) is H, —OH, (C₁-C₄)alkoxy, halo(C₁-C₄)alkoxy or (C₁-C₈)alkyl; and

R⁵ is H or (C₁-C₄)alkyl.

One skilled in the art will recognize that substituents and othermoieties of the compounds of the generic formulae herein should beselected in order to provide a compound which is sufficiently stable toprovide a pharmaceutically useful compound which can be formulated intoan acceptably stable pharmaceutical composition. Compounds which havesuch stability are contemplated as falling within the scope of thepresent invention. It should be understood by one skilled in the artthat any combination of the definitions and substituents described aboveshould not result in an inoperable species or compound.

Preparation of Macrocyclic Compounds

A compound of the present invention such as those of Formula I, I-a,I-b, I-c, I-d, I-e or II can be prepared according to the schemesdescribed below, but it shall be appreciated that modifications of theillustrated process or other process can also be used. As illustrated inScheme 1, the macrocyclic compounds M are synthesized from the five keycomponents A-E by combining them together in sequence with theappropriate use of protecting groups (PG¹-PG⁸) by one skilled in theart. The hashed lines numbered 1-5, hereby referred to as Connection 1,Connection 2, etc., respectively, are the 5 connections for combiningComponents A-E. The order in which the specific connections occur, canvary, and are dependent on the choice of protecting groups and chemistryrequired. In certain embodiments, Connections 3, 4 or 5 are used as thefinal macrocyclization step.

Illustratively, Connections 1 through 5 can be performed as describedbelow:

Connections 1, 2 and 3 are amide bonds. The connections are made betweenthe respective acid and amine using standard peptide coupling agents(EDC/HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMO, etc.) known to oneskilled in the art. The acid and amine coupling partners are combinedwith a coupling agent in an organic solvent, e.g., DMF, dichloromethane,acetonitrile, etc., in the presence of a base, e.g., DIPEA,triethylamine, etc., at RT or slightly elevated temperature. When any ofthese three steps are chosen as the final macrocyclization step, forexample Connection 3, then macrolactamization conditions are preferred.Suitable macrolactamization procedures include, but are not limited to,those found in the following reference: Davies, J. S. J. Peptide Sci.2003, 9, 471-501.

Connection 4 is typically a carbon-carbon bond or a heteroatom-carbonbond where the heteroatom is O, S or N. When Connection 4 is acarbon-carbon bond, then standard carbon-carbon bond forming procedurestypically involving metal mediated cross coupling reactions arepreferred. Preferably the carbon-carbon bond is formed using a Heck typecoupling between an sp2 halide group and an terminal alkene, a Suzukicoupling between an sp2 halide group and a vinyl or aryl boronate, or aring closing metathesis (RCM) between two alkenes. Stille reactions canalso be performed between a vinyl stannane and an aryl or vinyl halideas described in Journal of American Chemical Society 2000, 122, 3830Nicolaou et al. In each of the examples above the aryl or vinyl halidegroup can also be an aryl or vinyl triflate.

For example, when A²-Y² in A contains a terminal alkene, such as—CH═CH₂, and A¹-Y¹ in B contains a terminal alkene, or CH₃—CH═CH— then across metathesis reaction is performed. The two components are mixed insolvent, e.g., acetonitrile, toluene and a metathesis catalyst, e.g.,Grubbs I, Grubbs II or Hoyveda-Grubbs I, Hoyveda-Grubbs II is addedfollowed by heating. If this connection is the final procedure to closethe macrocyclic ring, RCM conditions are preferred (e.g., more diluteconditions to avoid dimerization). For relevant RCM conditions andexamples see Journal of American Chemical Society 2003, 125, 3849Sedrani et al and Journal of American Chemical Society 2000, 122, 3830Nicolaou et al. A typical RCM procedure includes heating (eitherconventionally or by microwave) of the acyclic precursor in a solventsuch as toluene, or 1,2-dichloroethane, in the presence of a RCMcatalyst, e.g., Grubbs I, Grubbs II or Hoyveda-Grubbs I, Hoyveda-GrubbsII.

Alternatively, when Connection 4 is made via a Heck coupling reaction,the vinyl or aryl halide, or the triflate A and the alkene component Bare mixed in a polar solvent, e.g., acetonitrile, or toluene in thepresence of a Palladium(II) catalyst, e.g., Palladium(OAc)₂, a phosphineligand, e.g., P(o-toluene)₃, P(t-butyl)₃, etc., and a base, e.g.,triethylamine. The reaction mixture is heated either conventionally orin a microwave reactor.

Alternatively, when Connection 4 is made via a Suzuki coupling reaction,the vinyl or aryl halide, or the triflate A and the vinyl or arylboronate B are mixed in a suitable solvent, e.g., cyclopentyl methylether, toluene, DMF, DME, etc., in the presence of a Palladium catalyst(e.g., Palladium(II)Cl₂(p-NMe₂Ph)₂ and K₃PO₄ ortetrakis(triphenylphosphine)palladium(0) and a base, such as potassiumcarbonate). The reaction mixture is heated either conventionally or in amicrowave reactor. It is also possible in such a coupling reaction toreverse the reactive functionalities on the two starting materials, suchthat A is an aryl or vinyl boronate and B contains a vinyl or arylhalide or triflate.

Alternatively, Connection 4 can be a carbon-oxygen bond and in this casetypical alkylation or nucleophilic aromatic substitution conditions canbe used between a hydroxyl group and an alkyl halide, or aryl (orheteroaryl) halide. The hydroxyl reagent is mixed with the alkyl orheteroaryl halide (preferably an iodide or bromide), in an inertsolvent, e.g., CPME, DMF, THF, etc, in the presence base, e.g., cesiumcarbonate, cesium hydroxide, sodium hydride, MaHMDS, etc.; and heated.

Alternatively, Connection 4 can be a carbon-nitrogen bond and in thiscase typical alkylation, nucleophilic aromatic substitution or Buchwaldconditions can be used between an amine group and an alkyl halide orheteroaryl halide. For example, the amine and the alkyl or heteroarylhalide are mixed and heated in an inert solvent, e.g., CPME, in thepresence base, e.g., cesium carbonate, sodium hydride, etc. Analternative procedure for the carbon-nitrogen connection is to perform areductive amination between an amine and a carbonyl compound. Typicallythe amine and aldehyde or ketone are mixed in an inert solvent, e.g.,THF, dioxane and treated after a period of time with sodium acetoxyborohydride or alternative reducing agent.

Connection 5 is typically an amide (X¹═—NH or substituted N), or ester(X¹=0) bond. When forming the amide bond, standard coupling proceduresas described for Connections 1-3 can be used. In some embodiments, thisis the final step in closing the macrocycle. As such, macrolactamizationtypes of coupling procedures are more effective. Suitablemacrolactamization procedures include, but are not limited to, thosefound in Davies, J. S. J. Peptide Sci. 2003, 9, 471-501.

When forming the ester bond, standard coupling reagents (e.g., EDC, DCC,PyBOP, HATU, COMO) can be used, or when this is the final step information of the macrocycle, macrolactonization procedures are preferred(e.g., Shina, Yamaguchi). An exeplary method for the macrolactonizationstep can be found in Journal of American Chemical Society 2002, 124,4257 Paquette et al or Chemical Reviews 2006, 106(3), 911-939.Typically, the acid and alcohol are mixed in a polar solvent, e.g., DMF,acetonitrile, etc, in the presence of the coupling agent and a base,e.g., DIPEA, DMAP.

The following general schemes provide general examples and sequences forconstructing the macrocyclic compound M from the common precursors A-E.

Compounds A-E can be deprotected (PG²-PG⁸) using conditions described inGreene and Wuts, Protective Groups in Organic Synthesis, John Wley andSons, Inc. to provide Compounds 1a-1f.

In many cases the optimal protecting groups and their deprotectionmethods are as follows. For Compound E, the typical protecting group PG¹for the acid is a methyl or trichloroethyl ester. The methyl andtrichloroethyl esters can be removed by base, e.g., LiOH in a polarsolvent, e.g., aqueous THF, etc. The trichloroethyl ester can also beremoved by treatment with zinc and ammonium acetate in a polar solvent,e.g., THF. Typically, PG² and PG⁴ are acid labile groups, e.g., BOC, andare deprotected using HCl in dioxane, or TMSOTf in dioxane,dichloromethane. Typically, PG³ and PG⁵ are ester groups, removed bytreatment with alkali metal hydroxide in aqueous THF or dioxane.Typically PG⁶ is an acid labile group, e.g., BOC, for an amine andremoved as described for PG², or a silyl ether for a hydroxyl group, andcan be removed by treatment with HF.pyridine of TBAF in an organicsolvent, e.g., dichloromethane. Typically PG⁸ is an amine protectinggroup, e.g., BOC, and is removed as described for PG² or a silyl etherfor a hydroxyl group, which can be removed as described for PG⁶, or anacetate protecting group, which can be removed with an alkali metalhydroxide in aqueous THF or dioxane.

Compound 1f is then coupled to acid 1a using the conditions describedabove for Connection 1 to produce compound 1g. Compound 1g is thendeprotected using conditions described in Greene and Wuts and coupled to1c to provide 1h using the conditions described above for Connection 2.An alternative sequence for generating 1h begins with coupling amine 1bto acid 1c using conditions as described for Connection 2 above, to form1i; deprotection of the protecting group PG³ in 1i using conditionsdescribed in Greene and Wuts, and finally coupling with amine 1f usingconditions described for Connection 1 above forms 1h.

Compound 1h is deprotected at PG⁶ using conditions described in Greeneand Wuts and the amine is then coupled to 1d using the conditionsdescribed above for Connection 3 to form 1j. Protecting group PG¹ inCompound 1j is then removed using conditions described in Greene andWuts, and the acid is then coupled to 1e using conditions described forConnection 5 to form the acyclic intermediate 1k. An alternativesequence to 1k is first deprotection of PG¹ and then coupling to 1e asdescribed for Connection 5; followed by deprotection of PG⁶ as describedin Greene and Wuts, followed by coupling to 1d using the conditionsdescribed for Connection 3 to form 1k. Acyclic intermediate 1k is thensubjected to the RCM as described above for Connection 4 to form themacrocycle M.

Compound A is coupled to Compound B using the conditions described abovefor Connection 4 to generate 2a. Compound 2a is then deprotected at PG⁷as described in Greene and Wuts to generate acid 2b. Acid 2b is thencoupled to the deprotected product of 1 h (prepared from 1h bydeprotection of PG⁶ described in Greene and Wuts) to generate theprecursor 2c. Deprotection of 2c is carried out using conditionsdescribed in Greene and Wuts, and then the product is cyclized using theconditions described above for macrolactamization or macrolactonizationin Connection 5, to provide Compound M.

Compound 2a is deprotected at PG⁸ as described in Greene and Wuts andabove in Connection 5 to generate 3a which is then coupled, usingconditions described above for Connection 5, to the deprotected productof 1 h (prepared from 1 h by deprotection of PG¹ described in Greene andWuts) to generate the precursor 3b. Deprotection of 3b is carried outusing conditions described in Greene and Wuts, and then cyclized usingthe conditions described above for macrolactamization ormacrolactonization in Connection 3, to provide Compound M.

For example, when M contains a C═C as a result of RCM, Compound M ismixed in a solvent such as ethanol, methanol, etc., in the presence ofpalladium on carbon catalyst under an atmosphere of hydrogen gas toprovide reduced Compound M1. The final macrocycle M from Schemes 4-6often contains protecting groups on side-chains that require furtherremoval to generate the final compound M. Protecting groups on theR^(4a), R^(4b), R^(3a), R^(3b), A¹, A² and/or X¹ are removed usingconditions described in Greene and Wuts to generate Compound M2. Anothertransformation is click chemistry to produce triazole M3. Thistransformation is performed by treating the alkyne or azide in M, insolvent (e.g., DMF) with an alkyne or azide as appropriate in thepresence of CuI to form M3.

Deprotected compound M2 can be further transformed after deprotection toadditional macrocycle M. For example, treatment of M2 containing an —OHwith an alkyl halide in the presence of a base, e.g., cesium carbonate,in a suitable solvent, e.g., DMF, acetonitrile, etc., forms alkylatedproduct M4. M2 containing a ketone group is treated in a suitablesolvent (e.g., DMF, methanol, etc.) with an amine followed by theaddition of sodium acetoxyborohydride to form amine product M5.

Many B components containing an acid or ester with a terminal alkene orCH₃—CH═C— or vinyl/aryl boronate groups are commercially available orare described in the literature and can be used in the above schemesdirectly. In addition, the following schemes are examples of methodsthat can be used to generate additional B components.

In Scheme 8, part 1, a protected acid is treated with a strongdeprotonating base, e.g., LDA in an inert solvent, e.g., THF, at −78° C.and HMPA. A pre-cooled solution of dichloroacteylene (prepared bytreatment of trichlorethene with potassium hydride and MeOH (catalytic)in THF) is then added to generate the chloro acetylene product. Thisproduct is then reduced, for example, by treatment with Cu in aceticacid and THF, to generate the alkyne, which is then further reduced tothe alkene, for example, by treatment of an alcoholic solution of thealkyne with a poisoned palladium reducing agent (e.g., Lindlar) in thepresence of hydrogen gas. Alternatively, the alkyne is treated withCp₂ZrHCl in dichloromethane in the presence of pinnacolborane to formthe vinyl boronate.

In Scheme 8, part 2, a beta-keto ester with alpha substitution isconverted to the vinyl triflate, for example, by treating a THF solutionof the beta-keto ester with a base, e.g., LDA, in THF at −78° C.,followed by addition of PhN(Tf)₂. The triflate product is then treatedwith pyridine at elevated temperature to form the alkyne. The alkyne isthen treated as described above in part 1 to generate the alkene orvinyl boronate products.

In Scheme 8, part 3, a chiral aldol reaction is used. An acyl group isfirst attached to a chiral auxiliary, e.g., Evans, Oppolzer sultam (seeJACS 1990, 112, p 2767), using the standard amide bond formationconditions as described above for Connection 1-3. The Oppolzer auxiliaryproduct is treated with the aldehyde of choice, TBDMSOTf and base, e.g.,triethylamine, in anhydrous solvent, e.g., dichloromethane. The Evansauxiliary is treated with base, e.g., LDA, KHMDS, DIPEA, in organicsolvent, e.g., THF, at −78° C. and the aldehyde of choice in thepresence of a Lewis acid, e.g., TiCl₄, SnCl₄, BF₃OEt₂. Protection of theresulting alcohol from the aldol reaction is performed as described inGreene and Wuts, or alternatively alkylation with an alkyl halide orMeerwein's reagent, i.e., treatment with trimethyloxoniumtetrafluoroborate in an inert solvent, e.g., dichloromethane, isperformed. The auxiliary is then removed using standard alkali metalhydroxide removal conditions, e.g., LiOH in THF, or LiOH and hydrogenperoxide in THF, to provide the free acids product.

In Scheme 8, part 4, an Evans auxiliary is allylated with an allylhalide as described in Synlett 2002, 12, 2039-2040. The product is thenisomerized by treatment with RhCl₃ in ethanol and then the auxiliaryremoved by base and peroxide, e.g., LiOH and H₂O₂ in THF/Water.Alternatively the auxiliary is directly removed by LiOH and H₂O₂ inTHF/Water to provide the terminal alkene.

In Scheme 8, part 5, a Homer Wadsworth Emmons reaction is used on analdehyde (containing a terminal alkene) to generate the alpha-betaunsaturated ester, which is then selectively reduced to the ester. Forexample, the phosphonate is treated with base, e.g., sodium hydride, inTHF at low temperature, followed by addition of the aldehyde and warmingto generate the unsaturated ester. The product is reduced by treatmentwith magnesium powder in methanol.

In Scheme 8, part 6, an alpha-beta unsaturated acid is converted to theunsaturated Evans auxiliary (see Organic Letters 2007, 9, p 1635) and istreated with an aldehyde to generate the corresponding alkene product.The hydroxyl group is then protected using methods described in Greeneand Wuts and then the auxiliary is removed by treatment with base andperoxide, e.g., LiOH and H₂O₂ in THF/Water. The hydroxyl can also bealkylated by as described above for aldol Scheme 8, part 3.

In Scheme 8, part 7, a ketone is transformed via the nitro olefin asdescribed in Angew. Chem. Int. Ed. 2006, 45 (46), 7736. The nitro olefinis then treated with vinyl magnesium bromide in an inert solvent, e.g.,THF, in the presence of a copper(I)salt, e.g., CuI and trimethylsilylchloride. The nitro alkyl product after addition of the vinyl group isthen converted to the acid by treating with sodium nitrite and aceticacid in an inert polar solvent, e.g., DMSO.

Several types of A are available commercially or described in theliterature where X¹ is O or NH and Y² is a halide or A²-Y² contains analkene. The schemes below describe additional general methods forgenerating A.

In Scheme 9, part 1 (Y² is a halogen in A); the starting Compound 6a istypically a commercially available aromatic compound, that containshalogen Y² and a group Z that can be transformed to the ketone 6b.Typical Z groups are halide, acid, or aldehyde, for example.

When Z is an acid, 6a is treated with a coupling agent, e.g., HATU, EDCin the presence of a base, e.g., DIPEA and the Weinreb amine (Me-NH—OMe)to form the Weinreb amide. The amide is then treated with a nucleophile,e.g., TMS-CF₃ to form the CF₃ substituted ketone 6b or with a Grignardagent, e.g., MeMgBr in a solvent, e.g., THF, at −78° C. to form themethyl ketone 6b.

When Z is a halogen, then the initial conversion, if required, to a morereactive halogen is performed by treatment with NaI and acetyl chloridein an inert solvent, e.g., acetonitrile. The halogen is then transformedto the ketone by a Stille reaction with an ethoxyvinyl stannane. Thehalide is treated in an inert solvent, e.g., toluene, with the stannaneand a palladium (II) catalyst, e.g., PdCl₂(PPh₃)₂, followed by treatmentof the product with 2M HCl to afford ketone 6b. In some cases theformation of an alkyl lithium reagent from the halide group can beperformed by treatment with nBuLi at −78° C. in THF and then addition ofa N-methoxy-N-methyl amide to afford the ketone 6b (e.g.,N-methoxy-N-methylacetamide affords the R⁵ is methyl ketone 6b). A finalmethod to generate the ketone 6b is through a vinyl group. 6a is treatedwith a vinyltrifluoroborate in the presence of a palladium catalyst,e.g., PdCl₂(dppf) and then the vinyl product is subsequently ozonolysedin a polar solvent, e.g., methanol at low temperature to give analdehyde. The aldehyde is then reacted with a nucleophile, e.g., TSM-CF₃or a Grignard reagent, e.g., MeMgBr to afford a secondary alcoholproduct. The secondary alcohol is then oxidized with Dess MartinPeriodinane to give the desired ketone 6b or can be used as A itself.

Chiral alcohol (X¹ is O) and amine (X¹ is NH) A are generated usingChiral reduction methods on the ketone 6b. Chiral alcohol 6c is formedfrom 6b using one of the numerous chiral reduction methods available inthe literature. Typically, dichloro(p-cumene)ruthenium(II) dimer and(1R,2R)-(−)-N-p-tosyl-1,2-diphenylethylenediamine are combined in water,and sodium formate and 6b is added in a water miscible solvent such astetrahydrofuran. The reaction is then stirred at a temperature betweenambient and reflux to produce 6c where X¹ is O. Alternatively, a chiralCBS reduction can be performed in an inert solvent, e.g., THF at lowtemperature to also afford the chiral alcohol 6c. Protection of the OHin 6c is performed using methods described in Greene and Wuts, typicallya TBS ether or acetyl group are used to provide A (X¹ is O).

Alternatively, to make chiral A (X¹ is NH), ketone 6b is first convertedto a chiral imine (R¹⁰ is chiral group) and then reduced using a varietyof methods described in the literature. For example, a chiralsulfinamide is reacted with the ketone 6b to afford a chiral sulfinimine6d, which is then reduced with a suitable reducing agent, typicallyNaBH₄, or selectride, or a Noyori type reduction as described for thechiral alcohol above, with dichloro(p-cumene)ruthenium(II) dimer and(1R,2R)-(−)-N-p-tosyl-1,2-diphenylethylenediamine. The sulfinamideauxiliary is then removed by treatment with mineral acid, preferably HClin a suitable organic solvent such as methanol, to afford 6e where X¹ isNH. Protection of the NH group can then be performed as described inGreene and Wuts to generate A (X¹ is NH).

In part 2 of Scheme 9, the synthesis of compound A where Y² is H andA²-Y² contains —CH═CH₂, a precursor for metathesis and cross couplingreactions is illustrated. Exemplary methods are as follows.

Compound 6c or 6e generated in Scheme 9, part 1 is first optionallyprotected on X¹ using a suitable protecting group as described in Greeneand Wuts, and then a vinyl group is introduced by a suitable crosscoupling method onto the aryl or sp2 halide. For example, a transitionmetal mediated coupling with a vinyl stannane or vinyl tetrafluoroborateusing a suitable palladium catalyst, e.g., PdCl₂(dppf)₂ or PdCl₂(PPh₃)₂in a suitable organic solvent, e.g., acetonitrile, dichloromethane,etc., with either thermal or microwave heating affords alkene A.

Another typical method that can be used to introduce a vinyl group isstarting from the ketone 6b where Y is OH. Initially, triflation of thealcohol is performed by treatment with Tf₂O in the presence of a base,e.g., pyridine. The ketone group is then reduced with a Noyorireduction, or as described above through the sulfonamide, to provide thechiral alcohol or amine. The chiral alcohol or amine is then protectedas described in Greene and Wuts, and then the triflate is reacted with avinyl cross coupling reagent, e.g., vinyl stannane in a Stille coupling,or a vinyltrifluoroborate as described above to introduce the alkene. Afurther example of alkene generation using 6b ketone is via introductionof an allyl group. Thus, 6b where Y is OH is treated in an inert solventin the presence of a suitable base, e.g., alkali metal carbonate,preferably potassium carbonate with allyl bromide to form 6f. Compound6f is then similar to ketone 6b and is therefore able to be transformedas described above in part 1 to Compound A where X¹ is O or NH withprotecting group PG⁸.

Pharmaceutical Formulations

The compounds of this invention are formulated with conventionalcarriers and excipients, which will be selected in accord with ordinarypractice. Tablets will contain excipients, glidants, fillers, bindersand the like. Aqueous formulations are prepared in sterile form, andwhen intended for delivery by other than oral administration generallywill be isotonic.

While it is possible for the active ingredients to be administered aloneit may be preferable to present them as pharmaceutical formulations. Theformulations of the invention, both for veterinary and for human use,comprise at least one active ingredient, together with one or moreacceptable carriers and optionally other therapeutic ingredients.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also beadministered as a bolus, electuary or paste.

The effective dose of an active ingredient depends at least on thenature of the condition being treated, toxicity, whether the compound isbeing used prophylactically (lower doses) or against an active viralinfection, the method of delivery, and the pharmaceutical formulation,and will be determined by the clinician using conventional doseescalation studies. The effective dose can be expected to be from about0.0001 to about 100 mg/kg body weight per day; typically, from about0.01 to about 10 mg/kg body weight per day; more typically, from about0.01 to about 5 mg/kg body weight per day; most typically, from about0.05 to about 0.5 mg/kg body weight per day. For example, the dailycandidate dose for an adult human of approximately 70 kg body weightwill range from 1 mg to 1000 mg, preferably between 5 mg and 500 mg, andmay take the form of single or multiple doses.

Combination Therapy

The compounds of the present invention may be combined with one or moreactive agents. Non-limiting examples of suitable active agents to becombined include one or more interferons, ribavirin or its analogs, HCVNS3 protease inhibitors, NS5a inhibitors, alpha-glucosidase 1inhibitors, hepatoprotectants, mevalonate decarboxylase antagonists,antagonists of the renin-angiotensin system, other anti-fibrotic agents,endothelin antagonists, nucleoside or nucleotide inhibitors of HCV NSSBpolymerase, non-nucleoside inhibitors of HCV NSSB polymerase, HCV NSSAinhibitors, TLR-7 agonists, cyclophillin inhibitors, HCV IRESinhibitors, pharmacokinetic enhancers and other drugs for treating HCV;or mixtures thereof.

More specifically, one or more compounds to be combined are selectedfrom the group consisting of

1) interferons, e.g., pegylated rIFN-alpha 2b (PEG-Intron), pegylatedrIFN-alpha 2a (Pegasys), rIFN-alpha 2b (Intron A), rIFN-alpha 2a(Roferon-A), interferon alpha (MOR-22, OPC-18, Alfaferone, Alfanative,Multiferon, subalin), interferon alfacon-1 (Infergen), interferonalpha-n1 (Wellferon), interferon alpha-n3 (Alferon), interferon-beta(Avonex, DL-8234), interferon-omega (omega DUROS, Biomed 510),albinterferon alpha-2b (Albuferon), IFN alpha XL, BLX-883 (Locteron),DA-3021, glycosylated interferon alpha-2b (AVI-005), PEG-Infergen,PEGylated interferon lambda (PEGylated IL-29), and belerofon;

2) ribavirin and its analogs, e.g., ribavirin (Rebetol, Copegus), andtaribavirin (Virami3);

3) HCV NS3 protease inhibitors, e.g., boceprevir (SCH-503034, SCH-7),telaprevir (VX-950), VX-813, TMC-435 (TMC435350), ABT-450, BI-201335,BI-1230, MK-7009, SCH-900518, VBY-376, VX-500, GS-9256, GS-9451,BMS-790052, BMS-605339, PHX-1766, AS-101, YH-5258, YH5530, YH5531, andITMN-191 (R-7227);

4) alpha-glucosidase 1 inhibitors, e.g., celgosivir (MX-3253), Miglitol,and UT-231B;

5) hepatoprotectants, e.g., emericasan (IDN-6556), ME-3738, GS-9450(LB-84451), silibilin, and MitoQ;

6) nucleoside or nucleotide inhibitors of HCV NS5B polymerase, e.g.,R1626, R7128 (R4048), IDX184, IDX-102, PSI-7851, BCX-4678,valopicitabine (NM-283), GS-6620 and MK-0608;

7) non-nucleoside inhibitors of HCV NS5B polymerase, e.g., filibuvir(PF-868554), ABT-333, ABT-072, BI-207127, VCH-759, VCH-916, JTK-652,MK-3281, VBY-708, VCH-222, A848837, ANA-598, GL60667, GL59728, A-63890,A-48773, A-48547, BC-2329, VCH-796 (nesbuvir), GSK625433, BILN-1941,XTL-2125, and GS-9190;

8) HCV NSSA inhibitors, e.g., AZD-2836 (A-831), AZD-7295 (A-689), andBMS-790052;

9) TLR-7 agonists, e.g., imiquimod, 852A, GS-9524, ANA-773, ANA-975,AZD-8848 (DSP-3025), PF-04878691, and SM-360320;

10) cyclophillin inhibitors, e.g., DEBIO-025, SCY-635, and NIM811;

11) HCV IRES inhibitors, e.g., MCI-067;

12) pharmacokinetic enhancers, e.g., BAS-100, SPI-452, PF-4194477,TMC-41629, GS-9350, GS-9585, and roxythromycin;

13) other drugs for treating HCV, e.g., thymosin alpha 1 (Zadaxin),nitazoxanide (Alinea, NTZ), BIVN-401 (virostat), PYN-17 (altirex),KPE02003002, actilon (CPG-10101), GS-9525, KRN-7000, civacir, GI-5005,XTL-6865, BIT225, PTX-111, ITX2865, TT-033i, ANA 971, NOc-205, tarvacin,EHC-18, VGX-410C, EMZ-702, AVI 4065, BMS-650032, BMS-791325,Bavituximab, MDX-1106 (ONO-4538), Oglufanide, FK-788, and VX-497(merimepodib);

14) mevalonate decarboxylase antagonists, e.g., statins, HMGCoA synthaseinhibitors (e.g., hymeglusin), squalene synthesis inhibitors (e.g.,zaragozic acid);

15) angiotensin II receptor antagonists, e.g., losartan, irbesartan,olmesartan, candesartan, valsartan, telmisartan, eprosartan;

16) angiotensin-converting enzyme inhibitors, e.g., captopril,zofenopril, enalapril, ramipril, quinapril, perindopril, lisinopril,benazepril, fosinopril;

17) other anti-fibrotic agents, e.g., amiloride; and

18) endothelin antagonists, e.g. bosentan and ambrisentan.

In yet another embodiment, the present application provides acombination therapy comprising a composition of the present inventionand a second pharmaceutical composition comprising at least oneadditional therapeutic agent selected from the group consisting of HIVprotease inhibiting compounds, HIV non-nucleoside inhibitors of reversetranscriptase, HIV nucleoside inhibitors of reverse transcriptase, HIVnucleotide inhibitors of reverse transcriptase, HIV integraseinhibitors, gp41 inhibitors, CXCR4 inhibitors, gp120 inhibitors, CCR5inhibitors, interferons, ribavirin analogs, NS3 protease inhibitors,NS5a inhibitors, alpha-glucosidase 1 inhibitors, cyclophilin inhibitors,hepatoprotectants, non-nucleoside inhibitors of HCV, and other drugs fortreating HCV, and combinations thereof.

More specifically, one or more compounds of the present invention may becombined with one or more compounds selected from the group consistingof 1) HIV protease inhibitors, e.g., amprenavir, atazanavir,fosamprenavir, indinavir, lopinavir, ritonavir, lopinavir+ritonavir,nelfinavir, saquinavir, tipranavir, brecanavir, darunavir, TMC-126,TMC-114, mozenavir (DMP-450), JE-2147 (AG1776), AG1859, DG35, L-756423,R00334649, KNI-272, DPC-681, DPC-684, and GW640385X, DG17, PPL-100, 2) aHIV non-nucleoside inhibitor of reverse transcriptase, e.g.,capravirine, emivirine, delaviridine, efavirenz, nevirapine, (+)calanolide A, etravirine, GW5634, DPC-083, DPC-961, DPC-963, Mb-150, andTMC-120, TMC-278 (rilpivirine), efavirenz, BILR 355 BS, VRX 840773,UK-453,061, RDEA806, 3) a HIV nucleoside inhibitor of reversetranscriptase, e.g., zidovudine, emtricitabine, didanosine, stavudine,zalcitabine, lamivudine, abacavir, amdoxovir, elvucitabine, alovudine,Mb-210, racivir (±-FTC), D-d4FC, emtricitabine, phosphazide, fozivudinetidoxil, fosalvudine tidoxil, apricitibine (AVX754), amdoxovir, KP-1461,abacavir+lamivudine, abacavir+lamivudine+zidovudine,zidovudine+lamivudine, 4) a HIV nucleotide inhibitor of reversetranscriptase, e.g., tenofovir, tenofovir disoproxilfumarate+emtricitabine, tenofovir disoproxilfumarate+emtricitabine+efavirenz, and adefovir, 5) a HIV integraseinhibitor, e.g., curcumin, derivatives of curcumin, chicoric acid,derivatives of chicoric acid, 3,5-dicaffeoylquinic acid, derivatives of3,5-dicaffeoylquinic acid, aurintricarboxylic acid, derivatives ofaurintricarboxylic acid, caffeic acid phenethyl ester, derivatives ofcaffeic acid phenethyl ester, tyrphostin, derivatives of tyrphostin,quercetin, derivatives of quercetin, S-1360, zintevir (AR-177),L-870812, and L-870810, MK-0518 (raltegravir), BMS-707035, MK-2048,BA-011, BMS-538158, GSK364735C, 6) a gp41 inhibitor, e.g., enfuvirtide,sifuvirtide, FB006M, TRI-1144, SPC3, DES6, Locus gp41, CovX, and REP 9,7) a CXCR4 inhibitor, e.g., AMD-070, 8) an entry inhibitor, e.g., SPO1A,TNX-355, 9) a gp120 inhibitor, e.g., BMS-488043 and BlockAide/CR, 10) aG6PD and NADH-oxidase inhibitor, e.g., immunitin, 10) a CCR5 inhibitor,e.g., aplaviroc, vicriviroc, INCB9471, PRO-140, INCB15050, PF-232798,CCR5mAb004, and maraviroc, 11) an interferon, e.g., pegylated rIFN-alpha2b, pegylated rIFN-alpha 2a, rIFN-alpha 2b, IFN alpha-2b XL, rIFN-alpha2a, consensus IFN alpha, infergen, rebif, locteron, AVI-005,PEG-infergen, pegylated IFN-beta, oral interferon alpha, feron,reaferon, intermax alpha, r-IFN-beta, infergen+actimmune, IFN-omega withDUROS, and albuferon, 12) ribavirin analogs, e.g., rebetol, copegus,VX-497, and viramidine (taribavirin) 13) NS5a inhibitors, e.g., A-831,A-689 and BMS-790052, 14) NS5b polymerase inhibitors, e.g., NM-283,valopicitabine, R1626, PSI-6130 (R1656), IDX184, PSI-7851, HCc-796, BILB1941, MK-0608, NM-107, R7128, VCH-759, PF-868554, GSK625433, andXTL-2125, 15) NS3 protease inhibitors, e.g., SCH-503034 (SCH-7), VX-950(Telaprevir), ITMN-191, and BILN-2065, 16) alpha-glucosidase 1inhibitors, e.g., MX-3253 (celgosivir) and UT-231B, 17)hepatoprotectants, e.g., IDN-6556, ME 3738, MitoQ, and LB-84451, 18)non-nucleoside inhibitors of HCV, e.g., benzimidazole derivatives,benzo-1,2,4-thiadiazine derivatives, and phenylalanine derivatives, 19)other drugs for treating HCV, e.g., zadaxin, nitazoxanide (alinea),BIVN-401 (virostat), DEBIO-025, VGX-410C, EMZ-702, AVI 4065,bavituximab, oglufanide, PYN-17, KPE02003002, actilon (CPG-10101),KRN-7000, civacir, GI-5005, ANA-975, XTL-6865, ANA 971, NOc-205,tarvacin, EHC-18, and NIM811, 19) pharmacokinetic enhancers, e.g.,BAS-100, SPI-452, PF-4194477, TMC-41629, GS-9350, GS-9585, androxythromycin, 20)RNAse H inhibitors, e.g., ODN-93 and ODN-112, 21)other anti-HIV agents, e.g., VGc-1, PA-457 (bevirimat), ampligen,HRG214, cytolin, polymun, VGX-410, KD247, AMZ 0026, CYT 99007, A-221HIV, BAY 50-4798, MDX010 (iplimumab), PBS119, ALG889, and PA-1050040.

In a specific aspect of this embodiment, the additional therapeuticagent is selected from ribavirin, telaprevir, boceprevir and sofosbuvir(GS-7977 (formerly PSI-7977)).

A combination therapy described herein may be administered as asimultaneous or sequential regimen. When administered sequentially, thecombination may be administered in two or more administrations.

Co-administration of a compound of the invention with one or more otheractive agents generally refers to simultaneous or sequentialadministration of a compound of the invention and one or more otheractive agents, such that therapeutically effective amounts of thecompound of the invention and one or more other active agents are bothpresent in the body of the patient.

Method for Treating Viral Infection

The present application provides a method for treating a Flaviviridaeviral infection comprising administering a therapeutically effectiveamount of a compound described herein or a pharmaceutically acceptablesalt, isotope, stereoisomer, mixture of stereoisomers, tautomer, esteror prodrug thereof, to a human subject in need thereof.

Also provided is a method for treating a Coronaviridae viral infectioncomprising administering a therapeutically effective amount of acompound described herein or a pharmaceutically acceptable salt,isotope, stereoisomer, mixture of stereoisomers, tautomer, ester orprodrug thereof, to a human subject in need thereof.

In one embodiment, the method of inhibiting or treating a diseasecomprises administering to an animal a composition comprising aneffective amount of one or more compounds of the invention or apharmaceutically acceptable salt, isotope, stereoisomer, mixture ofstereoisomers, tautomer, ester or prodrug thereof and a pharmaceuticallyacceptable carrier. The composition to be administered may furthercontain a secondary therapeutic agent as described above.

A method of the present application is particularly suitable for usewith humans, but may be used with other animals, particularly mammals,such as, for example, non-human primates, companion animals, farmanimals, laboratory animals, and wild and zoo animals.

A method of the present application is particularly useful to treatdiseases caused directly or indirectly by Flaviviridae virus since thecompounds of the present invention have inhibitory activity againstthose viruses. In some embodiments, therefore, a method of the presentinvention is used in inhibiting or treating diseases caused by aHepatitis C virus. In some embodiments, therefore, a method of thepresent invention is used in inhibiting or treating diseases caused by aHepatitis B virus. In an aspect, such a method is applied to a patientwith a disease caused by the viral infection such as dengue fever,yellow fever, hepatitis C, Japanese encephalitis, Kyasanur forestdisease, Murray valley encephalitis, St. Louis encephalitis, tick-borneencephalitis or West Nile encephalitis.

In some embodiments, a sustained virologic response is achieved at about12 weeks, at about 10 weeks, at about 8 weeks, at about 6 weeks, or atabout 4 weeks, or at about 4 months, or at about 5 months, or at about 6months, or at about 1 year, or at about 2 years.

A method of the present application is also particularly useful to treatdiseases caused directly or indirectly by Coronaviridae virus since thecompounds of the present invention have inhibitory activity againstthose viruses. In some embodiments, therefore, a method of the presentinvention is used in inhibiting or treating diseases caused by a SARScoronarirus. In an aspect, such a method is applied to a patient with adisease caused by the viral infection such as severe acute respiratorysyndrome (SARS), cancer, inflammation, obesity, acquired immunedeficiency syndrome (AIDS), or cirrhosis.

In another aspect, the compounds disclosed herein can be used fortreating cancer. In yet another aspect, the compounds disclosed hereincan be used for immunomodulation. In some embodiments, therefore, amethod of the present invention comprises adjusting an immune responseto a desired level, as in immunopotentiation, immunosuppression, orinduction of immunologic tolerance.

In some embodiments, the compound is administered for about 12 weeks. Infurther embodiments, the compound is administered for about 12 weeks orless, for about 10 weeks or less, for about 8 weeks or less, for about 6weeks or less, or for about 4 weeks or less. The compound may beadministered once daily, twice daily, once every other day, two times aweek, three times a week, four times a week, or five times a week.

In another aspect, the compounds disclosed herein can be used fortreating cancer. In yet another aspect, the compounds disclosed hereincan be used for immunomodulation. In some embodiments, therefore, amethod of the present invention comprises adjusting an immune responseto a desired level, as in immunopotentiation, immunosuppression, orinduction of immunologic tolerance.

EXAMPLES

The following examples are merely illustrative, and do not limit thisdisclosure in any way.

List of Abbreviations and Acronyms

Abbreviation Meaning ° C. Degree Celsius Ac Acetyl app Apparent AqAqueous Boc tert-Butoxycarbonyl Br Broad Bu Butyl cat Catalytic CBSCorey Bakshi Shibata CDMT 2-chloro-4,6-dimethoxy-1,3,5-triazine cmCentimeter COMU (1-Cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate CP/Cp Cyclopentyl CPMECyclopentyl methyl ether Cy/cHex Cyclohexyl d Doublet DBU1,8-Diazabicyclo[5.4.0]undec-7-ene DCC N,N′-dicyclohexylcarbodiimide DCEDichloroethane DCM Dichloromethane dd Doublet of doublets ddd Doublet ofdoublet of doublet ddt Doublet of doublet of triplet DIAD Diisopropylazodicarboxylate DIPEA N,N-Diisopropylethylamine DMAP4-Dimethylaminopyridine DME 1,2-dimethoxyethane DMF DimethylformamideDMSO Dimethylsulfoxide dppf 1,1′-bis(diphenylphosphino)ferrocene dqDoublet of quartet dt Doublet of triplet dtd Doublet of triplet ofdoublet EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Equiv/eqEquivalents Et Ethyl g Grams gen. Generation HATU(Dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methaniminium hexafluorophosphate HBTU2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphateHMDS Hexamethyldisilazane HMPA hexamethylphosphoramide HOBTHydroxybenzotriazole HPLC High-performance liquid chromatography hrs/hHours Hz Hertz IC₅₀ The half maximal inhibitory concentration Imimidazole i-Pr/iPr Isopropyl J Coupling constant Kg Kilogram LCMS Liquidchromatography-mass spectrometry LDA Lithium diisopropylamide M Molar mMultiplet m/z mass-to-charge ratio M+ Mass peak Me Methyl mg MilligramMHz Megahertz min Minute mL Milliliter mM Millimolar mm Millimeter mmolMillimole mol Mole Ms Methanesulfonyl MW Microwave N normal nM NanomolarNMM N-methylmorpholine NMR Nuclear magnetic resonance nPrOH n-propanolo-Tol o-Tolyl Ph Phenyl pTSA p-Toluenesulfonic acid Py/pyr PyridinePyAOP 7-azabenzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate PyBop benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate q Quartet Rf Retention factor RT/rt/r.t. Roomtemperature s Singlet sat. Saturated t Triplet TBAFTetra-n-butylammonium fluoride TBDMS/TBS tert-Butyldimethylsilyl TBDPStert-Butyldiphenylsilyl t-Bu tert-butyl td Triplet of doublets TEATriethylamine Tf Trifluoromethanesulfonyl TFA Trifluoroacetic acid THFTetrahydrofuran TLC Thin layer chromatography TMEDAtetramethylethylenediamine TMS Trimethylsilyl Tr/tr Retention time TsTosyl tt Triplet of triplet UV Ultraviolet wt. weight δ Chemical shiftμL Microliter μM Micromolar μmol Micromole

Example 1:(E)-(2R,5S,11S,14S,17R,18R)-18-Hydroxy-14-isopropyl-2,11,17-trimethyl-3,9,12,15,28-pentaaza-tricyclo[21.3.1.1*5,9]octacosa-1(26),21,23(27),24-tetraene-4,10,13,16-tetraone

A solution of1-((1R,5S)-10,10-dimethyl-3,3-dioxo-3lambda*6*-thia-4-aza-tricyclo[5.2.1.0*1,5*]dec-4-yl)-propan-1-one(3.95 g, 14.55 mmol) in toluene (50 mL) was prepared, then evaporated todryness. This process was repeated and the resulting white solid wasdissolved in anhydrous dichloromethane (16 mL). A small quantity ofcalcium hydride was added before adding tert-butyldimethylsilyltrifluoromethanesulfonate (3.83 mL, 14.5 mmol) and anhydroustriethylamine (2.33 mL, 16.7 mmol). The reaction mixture was stirred atRT (“RT”) under a nitrogen atmosphere for 15 hours (“h”). The resultingsolution was evaporated to yield a thick paste, which was re-dissolvedin anhydrous dichloromethane (15 mL) and added dropwise to a stirredsolution of 4-pentenal (2.69 g, 32.0 mmol) and titanium tetrachloride (1M in dichloromethane, 32 mL, 32 mmol) in anhydrous dichloromethane (20mL) at −78° C., under a nitrogen atmosphere. The reaction was stirred at−78° C. for 30 minutes (“min”) before diluting with saturated aqueousammonium chloride solution (100 mL). The layers were separated and theaqueous layer was extracted with dichloromethane (2×50 mL). The combinedextract was dried over sodium sulfate, filtered and evaporated to give abrown gum. This was purified by silica gel chromatography usingiso-hexanes/ethyl acetate 4:1 to yield the title compound (3.09 g, 60%)as a colorless gum.

A solution of 1a (12.0 g, 0.034 mol) in anhydrous dichloromethane (520mL) was cooled to 0° C., before adding pyridine (5.5 mL, 0.068 mol) thentert-butyldimethylsilyl trifluoromethanesulfonate (9 mL, 0.039 mol). Thereaction mixture was stirred at 0° C. for 15 min then allowed to warm toRT and stirred for a further 1.5 h. The reaction mixture was washed withsaturated sodium bicarbonate (400 mL). The aqueous wash wasback-extracted with dichloromethane (200 mL). The organic layers werecombined and washed with dilute brine (200 mL) and 2 M hydrochloric acid(200 mL). The solution was dried over sodium sulfate, filtered andevaporated to give the title product (15.29 g, 96%) as a white solid.

A solution of 1b (15.29 g, 0.0325 mol) in tetrahydrofuran (300 mL) wasprepared and a 2 M aqueous solution of lithium hydroxide (120 mL) wasadded. The stirred mixture was heated to 60° C. for 15 h. The reactionwas diluted with 2 M hydrochloric acid (250 mL). The layers wereseparated and the aqueous was extracted with ethyl acetate (2×200 mL).The organic layers were combined, dried over sodium sulfate, filteredand evaporated to give a cream solid (16.7 g). The solid was purified bysilica gel chromatography using 3:7 ethyl acetate/iso-hexanes to yieldthe title product (7.18 g, 81%) as a colorless gum.

A solution of (S)-2-tert-butoxycarbonylamino-propionic acid (3.28 g,17.32 mmol) in acetonitrile (160 mL) was cooled to 0° C. before additionof N,N-diisopropylethylamine (12 mL, 69.3 mmol) then2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(6.57 g, 17.32 mmol). The reaction mixture was stirred at 0° C. for 20min and a solution of (S)-hexahydropyridazine-3-carboxylic acid2,2,2-trichloroethyl ester trifluoroacetic acid salt (preparationdescribed in Angew. Chem. Int. Ed. Engl. 1999, 38, 2443, 6.49 g, 17.3mmol) in acetonitrile (80 mL) was added. The reaction was allowed towarm to RT and was stirred for 15 h. The reaction mixture was evaporatedthen re-dissolved in ethyl acetate (150 mL). The solution was washedwith brine (150 mL). The brine was back extracted with ethyl acetate (50mL). The organic layers were combined, dried over sodium sulfate,filtered and evaporated to give a dark oil. The oil was purified bysilica gel chromatography using iso-hexanes/ethyl acetate 1:1 to yieldthe title compound (6.88 g, 92%) as a colorless gum.

A solution of 1d (6.88 g, 15.9 mmol) in dichloromethane (200 mL) wasprepared and trifluoroacetic acid (50 mL) was added. The reactionmixture was stirred at RT for 2 h. TLC showed the reaction to becomplete. The solution was evaporated to give a brown oil. This wasazeotroped with toluene (50 mL) and the resultant oil was dried undervacuum to give(S)-1-((S)-2-amino-propionyl)-hexahydro-pyridazine-3-carboxylic acid2,2,2-trichloro-ethyl ester trifluoroacetic acid salt (7.8 g) as a browngum. A solution of ((S)-1-carbamoyl-2-methylpropyl)-carbamic acidtert-butyl ester in acetonitrile (300 mL) was cooled to 0° C. beforeadding N,N-diisopropylethylamine (13.8 mL, 79.7 mmol) and2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(6.33 g, 16.7 mmol). The reaction was stirred at 0° C. for 15 min beforeadding a solution of the(S)-1-((S)-2-amino-propionyl)-hexahydro-pyridazine-3-carboxylic acid2,2,2-trichloro-ethyl ester trifluoroacetic acid salt (ca. 15.9 mmol) inacetonitrile (85 mL). The reaction was stirred at 0° C. for a further 20min then allowed to warm to RT and stirred for 15 h. The reactionmixture was evaporated then re-dissolved in ethyl acetate (250 mL). Thesolution was washed with water (150 mL) then dried over sodium sulfate,filtered and evaporated to give a red oil. This was purified by silicagel chromatography using iso-hexanes/ethyl acetate 7:3 theniso-hexanes/ethyl acetate 1:1 to yield the title compound (8.2 g, 92%)as a pale orange amorphous solid.

A solution of 1e (10.0 g, 18.5 mmol) in anhydrous dichloromethane (200mL) was prepared and trimethylsilyl trifluoromethanesulfonate (5 mL,27.75 mmol) was added. The reaction mixture was stirred at RT for 2 h,then N,N-diisopropylethylamine (13.2 mL, 75.8 mmol) was added and thereaction mixture was evaporated to dryness. The residue was re-dissolvedin acetonitrile (200 mL) and a solution of(2R,3R)-3-(tert-butyl-dimethyl-silanyloxy)-2-methyl-hept-6-enoic acid(5.04 g, 18.5 mmol) in acetonitrile (60 mL) was added followed by1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (5.0 g, 26.4 mmol) and1-hydroxybenzotriazole (4.4 g, 26.4 mmol). The reaction mixture wasstirred at RT for 15 h. It was evaporated to give a thick yellow oil.The oil was purified by silica gel chromatography using 1:1 ethylacetate/iso-hexanes then 3:2 ethyl acetate/iso-hexanes to yield thetitle product (8.75 g, 69%) as a white solid.

A solution of (R)-bromo-α-methylbenzylamine (1.023 g, 5.112 mmol) indichloromethane (20 mL) was subsequently treated with triethylamine (720μL, 5.112 mmol) and di-tert-butyl dicarbonate (1.784 g, 8.179 mmol).After overnight stirring at RT, the volatiles were removed in vacuo andthe residue was purified by silica gel chromatography using a 50 gIsolute cartridge eluted with a continuous gradient ofiso-hexanes/diethyl ether 1:0 to 4:1 to afford the title compound (1.552g, 100%) as a white solid.

A solution of 1g (10.26 g, 0.0342 mol.) and tributyl(vinyl)tin (32.5 g,30 mL, 0.103 mol.) in toluene (175 mL) was purged with nitrogen for 30min before addition of bis(triphenylphosphine) palladium (II) dichloride(2.38 g, 0.0034 mol.). The stirred mixture was heated to 60° C. for 16 hbefore cooling to RT. The reaction mixture was filtered throughhyflo-supercel then evaporated to give a dark coloured oil. The oil waspurified by silica gel chromatography using iso-hexanes/ethyl acetate19:1 to yield the title compound (6.95 g, 82%) as a yellow oil.

A solution of 1h (6.95 g, 28.1 mmol.) in 1,4-dioxane (30 mL) wasprepared and a solution of hydrogen chloride in 1,4 dioxane (4 M, 60 mL)was added. The reaction mixture was stirred at RT for 2 h thenevaporated to dryness. The resultant solid was re-dissolved in tolueneand evaporated. The solid was triturated with diethyl ether, which wasremoved by decanting. The solid was then dried under vacuum to give thetitle compound (4.96 g, 96%) as an off-white solid.

A solution of 1f (8.75 g, 12.75 mmol) in tetrahydrofuran (350 mL) wasprepared and zinc powder (8.44 g, 127.5 mmol) was added followed by a 1M aqueous solution of ammonium acetate (90 mL, 90 mmol). The reactionmixture was vigorously stirred for 16 h then filtered throughhyflo-supercel. The solution was cooled over an ice bath before additionof aqueous ammonium chloride solution (350 mL). It was allowed tore-cool before acidifying to pH 1 by addition of 2 M hydrochloric acid.The layers were separated and the aqueous was extracted with ethylacetate (2×250 mL). The organic layers were combined, dried over sodiumsulfate filtered and evaporated to give a white solid. The solid wasazeotroped with toluene (3×200 mL) then dried under vacuum to give awhite solid (6.16 g), which was dissolved in acetonitrile (400 mL)before adding (R)-1-(3-vinyl-phenyl)-ethylamine hydrochloride (2.34 g,12.75 mmol) followed by N,N-diisopropylethylamine (8.9 mL, 51 mmol) and2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (6.8 g, 17.85 mmol). The reactionmixture was stirred at RT for 16 h and then evaporated to give a browngum. The gum was purified by silica gel chromatography using ethylacetate then 1:4 acetone/ethyl acetate to yield the title compound (5.51g, 76%) as a cream solid.

A solution of 1j (0.50 g, 0.88 mmol) in anhydrous dichloromethane (20mL) was cooled over an ice bath, before adding pyridine (92 μL, 1.14mmol) and tert-butyldimethylsilyl trifluoromethanesulfonate (242 μL,1.05 mmol). The reaction mixture was stirred at 0° C. for 15 min, thenallowed to warm to RT and stirred for 1 hour. The reaction mixture wasevaporated and the residue was purified by silica gel chromatographyusing ethyl acetate to yield the title product (477 mg, 80%) as a whitesolid.

A solution of 1k (477 mg, 0.7 mmol) in 1,2-dichloroethane (250 mL) wasprepared and Hoveyda-Grubbs 2^(nd) generation catalyst (43 mg, 0.07mmol) was added. The stirred reaction mixture was heated to 80° C. for 1hour. The reaction mixture was cooled to RT before adding silica gel.The mixture was stirred for 10 min then evaporated and the residue waspurified by silica gel chromatography using ethyl acetate to yield thetitle product (198 mg, 43%) as a white solid.

A solution of 1l (198 mg, 0.3 mmol) in tetrahydrofuran (20 mL) wascooled over an ice bath before adding a 1 M solution oftetrabutylammonium fluoride in tetrahydrofuran (1.5 mL, 1.5 mmol). Thereaction was allowed to warm to RT and was stirred for 1 hour. Thereaction mixture was treated with saturated aqueous sodium bicarbonatesolution (20 mL) and extracted with ethyl acetate (2×20 mL). The extractwas dried over sodium sulfate, filtered and evaporated. The residue waspurified by silica gel chromatography using 1:9 acetone/ethyl acetate toyield the title product (150 mg, 92%) as a white solid. ¹H NMR (300 MHz,d₆-DMSO) δ 0.83 (d, J=6.5 Hz, 3H), 0.85 (d, J=6.5 Hz, 3H), 1.12-1.21 (m,4H), 1.28 (d, J=7.1 Hz, 3H), 1.36 (d, J=6.9 Hz, 3H), 1.40-1.90 (m, 7H),1.92-2.08 (m, 1H), 2.19-2.34 (m, 2H), 2.67-2.80 (m, 1H), 3.56-3.65 (m,1H), 3.99-4.12 (m, 1H), 4.22 (br d, J=12.2 Hz, 1H), 4.75 (d, J=11.8 Hz,1H), 4.87-4.99 (m, 1H), 5.12-5.24 (m, 1H), 5.40 (d, J=4.5 Hz, 1H),6.14-6.33 (m, 2H), 7.10-7.35 (m, 5H), 7.88 (d, J=8.3 Hz, 1H), 8.55 (d,J=8.3 Hz, 1H). LCMS (m/z) 542.3 [M+H], Tr=1.87 min.

Example 2: (E)-(2R,5S,11S,14S,17R,18R)-14-Isopropyl-2,11,17-trimethyl-18-(2,2,2-trifluoro-ethoxy)-3,9,12,15,28-pentaaza-tricyclo[21.3.1.1*5,9]octacosa-1(26),21,23(27),24-tetraene-4,10,13,16-tetraone

A solution of hexamethyldisilazane (235 μL, 1.13 mmol) in anhydroustetrahydrofuran (4 mL) was cooled to −10° C. before adding a 2.5 Msolution of n-butyllithium (380 μL, 0.94 mmol). The stirred mixture waswarmed to 0° C. for 10 min before cooling to −78° C. A solution ofCompound 1 (102 mg, 0.188 mmol) in anhydrous dimethylformamide (1.5 mL)and anhydrous tetrahydrofuran (1.5 mL) was added dropwise over 2 minfollowed by the addition of 2,2,2-trifluoroethyltrifluoromethanesulfonate (135 μL, 0.94 mmol). The reaction mixture wasstirred at −78° C. under a nitrogen atmosphere then allowed to graduallywarm to RT. The reaction mixture was quenched with the addition of asaturated aqueous solution of ammonium chloride (15 mL). The mixture wasextracted with ethyl acetate (2×15 mL). The extract was dried oversodium sulfate, filtered and evaporated. The residue was purified bysilica gel chromatography using 1:9 acetone/ethyl acetate to give acolorless gum (65 mg). The gum was further purified by reverse phasechromatography, using a 10 g C18 cartridge eluted with 2:3acetonitrile/water. The partially evaporated fractions were extractedwith ethyl acetate (2×15 mL). The extract was dried over sodium sulfate,filtered and evaporated to yield the title product (17 mg, 30%) as awhite solid. ¹H NMR (300 MHz, CDCl₃) 0.92 (d, J=6.7 Hz, 3H), 0.96 (d,J=6.9 Hz, 3H), 1.32 (d, J=7.1 Hz, 3H), 1.39 (d, J=7.1 Hz, 3H), 1.50 (d,J=6.7 Hz, 3H), 1.74-1.85 (m, 1H), 1.86-2.08 (m, 3H), 2.10-2.40 (m, 3H),2.57-2.78 (m, 2H), 3.19-3.31 (m, 1H), 3.49 (app t, J=6.7 Hz, 1H),3.58-3.79 (m, 4H), 3.81-4.18 (m, 2H), 4.49 (br d, J=12.3 Hz, 1H),5.03-5.17 (m, 1H), 5.32-5.47 (m, 1H), 6.14-6.30 (m, 1H), 6.37-6.72 (m,4H), 7.06-7.30 (m, 4H). LCMS (m/z) 624.3 [M+H], Tr=2.57 min.

Example 3:(2R,5S,11S,14S,17R,18R)-18-Hydroxy-14-isopropyl-2,11,17-trimethyl-3,9,12,15,28-pentaaza-tricyclo[21.3.1.1*5,9]octacosa-1(26),23(27),24-triene-4,10,13,16-tetraone

A solution of Compound 1 (100 mg, 0.184 mmol) in methanol (5 mL) wasprepared and 10% palladium on carbon (5 mg) was added. The stirredmixture was placed under a hydrogen atmosphere for 1.5 h. The reactionmixture was filtered through hyflo-supercel then through a 0.2 μm filterbefore evaporating to give the title compound (95 mg, 95%) as a whitesolid. ¹H NMR (300 MHz, CDCl₃) 0.93 (d, J=7.1 Hz, 3H), 0.96 (d, J=7.1Hz, 3H), 1.32 (d, J=6.7 Hz, 3H), 1.37-2.16 (m, 12H), 1.54 (d, J=6.9 Hz,3H), 2.34-2.46 (m, 1H), 2.52-2.75 (m, 3H), 3.16-3.27 (m, 1H), 3.43-3.62(m, 3H), 3.74 (d, J=11.8 Hz, 1H), 3.98-4.06 (m, 1H), 4.50 (d, J=13.2 Hz,1H), 5.11-5.36 (m, 2H), 6.39-6.50 (m, 2H), 7.70 (d, J=8.5 Hz, 1H),7.06-7.28 (m, 4H). LCMS (m/z) 544.3 [M+H], Tr=1.91 min.

Example 4

To a suspension of sodium hydride (60% in mineral oil, 783 mg, 19.6mmol) in dry tetrahydrofuran at 0° C., under nitrogen was added dropwisetriethyl phosphonoacetate (3.38 mL, 19.6 mmol). The white suspension wasstirred for 1 h where it became a solution, then 2,2-dimethyl-4-pentenal(2.42 mL, 17.8 mmol) was slowly added and the resulting green/yellowsolution was stirred at 0° C. and allowed to warm to RT. After 72 hethanol (1 mL) was added to the solution, followed by water (100 mL) andthe organics were extracted with diethyl ether (2×200 mL). The combinedorganics were washed with water (200 mL), dried over magnesium sulfate,filtered and concentrated in vacuo to give a crude residue. This waspurified by silica gel chromatography, using iso-hexanes (144 mL), theniso-hexanes/diethyl ether 50:50 (72 mL), then diethyl ether (48 mL) togive the title compound (3.20 g, 99%) as an oil.

To 4a (3.20 g, 17.5 mmol) in dry methanol at RT, under nitrogen wasslowly added magnesium powder (1.28 g, 52.7 mmol) while monitoring thetemperature rise. After the addition was complete the mixture wasstirred at RT overnight. After this time, to the reaction was addedadditional magnesium powder (852 mg, 35.1 mmol) and the reaction mixturestirred for 2 h. The mixture was neutralized from pH 10 to pH 7 with 2 Mhydrochloric acid and then concentrated in vacuo to give a residue. Theresidue was suspended in ethyl acetate (300 mL) and water (500 mL) andthe layers separated. The aqueous phase was then re-extracted with ethylacetate (200 mL) and the combined organics were dried over magnesiumsulfate, filtered and concentrated in vacuo to give the title compound(1.96 g, 61%) as a colorless oil.

To 4b (2.02 g, 11.0 mmol) in a mixture of tetrahydrofuran (35 mL),methanol (9 mL) and water (9 mL) at RT, was added lithium hydroxidemonohydrate (1.38 g, 32.9 mmol) and the mixture was stirred at RT for 1h. After this time additional lithium hydroxide monohydrate (460 mg,11.0 mmol) was added and the reaction mixture stirred for 1 h. Afterthis time more lithium hydroxide monohydrate (460 mg, 11.0 mmol) wasadded and the reaction mixture stirred for 30 min. The reaction mixturewas then concentrated in vacuo and the residue diluted with water (200mL) and the organics extracted with diethyl ether (3×50 mL). The aqueousphase was acidified from pH 14 to pH 1 with 2 M hydrochloric acid andthe organics extracted with ethyl acetate (3×50 mL). The combined ethylacetate organics were then dried over sodium sulfate, filtered andconcentrated in vacuo to give the desired product (1.03 g, 60%) as anoil.

A solution of 1e (1.06 g, 2.00 mmol) in dichloromethane (10 mL) wascooled in an ice-water bath under nitrogen. Trimethylsilyltrifluoromethanesulfonate (0.591 mL, 4.00 mmol) was added dropwise, andthe resulting solution was stirred for 1.5 h. The reaction was quenchedwith N,N-diisopropylethylamine (1.4 mL, 8.00 mmol) and the reactionmixture was concentrated in vacuo to give(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester a white solid which was used withoutfurther purification. To(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester generated in the previous step wasadded a solution of 4,4-dimethyl-hept-6-enoic acid (343 mg, 2.20 mmol)in acetonitrile (70 mL). To this mixture was addedN,N-diisopropylethylamine (1.4 mL, 8.03 mmol) and the reaction cooled inan ice-water bath before the addition of2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate methanaminium (1.07 g, 2.80 mmol). The yellowsolution was then stirred and allowed to warm to RT overnight. Thereaction mixture was quenched with 2 M hydrochloric acid (40 mL) andconcentration in vacuo. To the residue was added water (200 mL) and theorganics were extracted with ethyl acetate (3×150 mL). The combinedorganics were then washed with brine (100 mL) and sodium hydrogencarbonate (200 mL). The organics were then dried over magnesium sulfate,filtered and concentrated in vacuo to give a crude yellow oil (2.27 g).This was purified by silica gel chromatography using iso-hexanes/ethylacetate 90:10 (300 mL) then iso-hexanes/acetone 50:50 (300 mL) theniso-hexanes/acetone 0:100 to give the title compound (1.10 g, 97%) as acolorless oil.

A solution of 4d (1.10 g, 1.94 mmol) in tetrahydrofuran (40 mL) wasprepared and zinc powder (2.79 g, 42.6 mmol) was added followed by asolution of ammonium acetate (2.24 g, 29.0 mmol) in water (10 mL). Thereaction mixture was stirred at RT for 24 h. Saturated aqueous potassiumhydrogen sulphate (pH 2, 30 mL) and ethyl acetate (50 mL) were added andthe suspension filtered through hyflo-supercel washing through withethyl acetate. The layers were separated. And the aqueous phase wasextracted with ethyl acetate (3×100 mL). The combined organic layerswere washed with brine (250 mL), dried over sodium sulfate, filtered andevaporated to give a colorless gum. The residue was azeotroped withtoluene (3×100 mL) to give the title compound (816 mg, 96%) as a whitesolid.

4e (439 mg, 1.00 mmol) was suspended in acetonitrile (35 mL) and(R)-1-(3-vinyl-phenyl)-ethylamine hydrochloride (202 mg, 1.10 mmol) wasadded followed by N,N-diisopropylethylamine (700 μL, 5.00 mmol) and themixture cooled in an ice-water bath before addition of2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate methanaminium (532 mg, 1.40 mmol). The reactionmixture was stirred and allowed to warm to RT over 72 h. To the reactionmixture was added 2 M hydrochloric acid (20 mL) and the mixtureconcentrated in vacuo to give a residue. The aqueous layer was extractedwith ethyl acetate (3×100 mL) and the combined ethyl acetate layers werewashed with brine followed by sodium hydrogen carbonate (100 mL) thendried over sodium sulfate, filtered and concentrated in vacuo to give acrude residue. The residue was purified by silica gel chromatographyusing iso-hexanes/ethyl acetate 50:50 then neat ethyl acetate to givethe title compound (402 mg, 71%).

To a solution of 4f (402 mg, 0.71 mmol) in 1,2-dichloroethane (210 mL)was added Hoveyda-Grubbs 2^(nd) generation catalyst (45 mg, 0.07 mmol)and the reaction mixture heated at 80° C. for 1 h. After this time themixture was allowed to cool to RT and concentrated in vacuo. The residuewas purified by silica gel chromatography using a gradient of ethylacetate to ethyl acetate/acetone 75:25. Impure product (240 mg) wascollected which was further purified by silica gel chromatography usingethyl acetate. After repurification, product (43 mg) was collectedcontaining minor impurities. The impurities were removed via triturationfrom diethyl ether to give the title compound (30 mg, 8%) as a whitesolid. ¹H NMR (300 MHz, CD₃OD) 0.89 (d, J=6.7 Hz, 3H), 0.92 (d, J=6.7Hz, 3H), 0.96 (s, 3H), 0.97 (s, 3H), 1.35 (d, J=6.9 Hz, 3H), 1.50 (d,J=7.1 Hz, 3H), 1.59-1.83 (m, 3H), 1.85-2.00 (m, 3H), 2.09 (d, J=7.1 Hz,2H), 2.15-2.25 (m, 1H), 2.31-2.44 (m, 1H), 2.78 (td, J=2.7, 12.7 Hz,1H), 3.40-3.72 (m, 2H), 4.05-4.17 (m, 1H), 4.34 (brd, J=13.2 Hz, 1H),5.05 (q, J=7.1 Hz, 1H), 5.35 (q, J=7.1 Hz, 1H), 6.19-6.32 (m, 1H), 6.37(d, J=15.8 Hz, 1H), 7.15 (d, J=7.6 Hz, 1H), 7.21-7.29 (m, 2H), 7.37 (d,J=7.8 Hz, 1H). LCMS (m/z) 540.3 [M+H], Tr=2.41 min.

Example 5

A solution of 6-bromo-3-chloro-isoquinoline (8.0 g, 33 mmol) andtributyl-(1-ethoxyvinyl)-tin (14.88 g, 14 mL, 41.2 mmol) in toluene (100mL) was degassed with nitrogen for 30 min.Bis(triphenylphosphine)palladium(II) dichloride (1.16 g, 1.65 mmol, 5mol %) was added and the reaction mixture was heated at 60° C. for 20 h.The reaction mixture was cooled to RT, the mixture was filtered and thefiltrate was evaporated. The residue was purified by silica gelchromatography using a gradient of iso-hexanes/ethyl acetate 20:1 to10:1 to afford the title compound (7.1 g, 92%) as a yellow solid.

A solution of 5a (7.1 g, 30 mmol) in 1,4-dioxane (60 mL) and 2 Mhydrochloric acid (30 mL) was stirred at RT for 30 min. The majority ofthe solvent was evaporated and the residue was partitioned between ethylacetate and water. The organic extracts were combined, washed with waterand brine, dried over sodium sulfate, filtered and evaporated. Theresidue was triturated with 5% ether in iso-hexanes and the resultingsolid was collected and dried to afford the title compound (6.0 g, 97%)as a white solid.

A solution of 5b (1.72 g, 8.3 mmol) in tetrahydrofuran (40 mL) wasstirred under nitrogen. Titanium (IV) ethoxide (3.8 g, 3.45 mL, 16.6mmol, tech. grade) was added followed by(R)-(+)-2-methyl-propanesulfinimide (1.11 g, 9.2 mmol) and the reactionmixture was stirred at 60° C. under nitrogen for 18 h. Additional(R)-(+)-2-methyl-propanesulfinimide (190 mg, 1.67 mmol) was added andthe reaction mixture was stirred at 65° C. for further 2 h. The reactionmixture was cooled to RT and ethyl acetate and brine were added. Thesuspension was filtered through Celite and the filter pad was washedwith ethyl acetate. The ethyl acetate layer was separated, washed withbrine, dried over sodium sulfate, filtered and evaporated. The residuewas purified by silica gel chromatography using a gradient ofiso-hexanes/ethyl acetate 7:3 to 3:7 to afford the title compound (2.2g, 86%) as a yellow solid.

A mixture of (1S,2R)-(−)-cis-1-amino-2-indanol (60 mg, 0.4 mmol),dichloro (p-cymene) ruthenium(II) dimer (122 mg, 0.2 mmol) and powdered4 {acute over (Å)} molecular sieves (2 g) was suspended in anhydrous2-propanol (9 mL) and stirred under nitrogen. The suspension was heatedat 90° C. for 20 min. The reaction mixture was cooled to 40° C. and asolution of 5c (1.23 g, 4 mmol) in 2-propanol (28 mL) was added followedby a solution of potassium tert-butoxide (122 mg, 1.1 mmol) in2-propanol (10 mL). The reaction mixture was stirred for 2 h at 40° C.and then allowed to cool. The mixture was poured directly onto a silicagel cartridge and eluted with ethyl acetate to give, after evaporation,the title compound (1.19 g, 96%) as a brown gum.

A mixture of 5d (0.66 g, 2.11 mmol), tributyl(vinyl)tin (1.85 mL, 6.35mmol) and palladium tetrakis(triphenylphosphine) (488 mg, 0.42 mmol) in1,4-dioxane (10.5 mL) was capped in a microwave vial. The reactionmixture was irradiated and stirred at 160° C. for 40 min in a microwavereactor. A second reaction was carried under identical scale andconditions and the reaction mixtures combined and evaporated. Theresidue was purified by silica gel chromatography using a gradient of1:3 to 1:0 ethyl acetate/iso-hexanes to afford the title compound (1 g)as a brown gum. ¹H NMR (300 MHz, CDCl₃) δ1.27 (s, 9H), 1.62 (d, J=7.7Hz, 3H), 3.53 (br s, 1H), 4.69-4.77 (m, 1H), 5.51 (dd, J=10.7, 1.3 Hz,1H), 6.39 (dd, J=17.4, 1.3 Hz, 1H), 6.95 (dd, J=17.2, 10.7 Hz, 1H),7.55-7.58 (m, 2H), 7.75 (s, 1H), 7.95 (d, J=8.3 Hz, 1H), 9.20 (s, 1H).LCMS (m/z) 303.0 [M+H], Tr=1.48 min.

5e was suspended in 4 M hydrochloric acid in 1,4-dioxane (17 mL, 68mmol) and methanol was added (34 mL). The reaction mixture was stirredfor 90 min and then evaporated. The residue was passed through a SCXcartridge eluting with methanol and then methanolic ammonia. The basicfraction was collected and evaporated to give the title compound (530mg, 63% over 2 steps) as a beige solid.

A solution of1-((1R,5S)-10,10-dimethyl-3,3-dioxo-3lambda*6*-thia-4-aza-tricyclo[5.2.1.0*1,5*]dec-4-yl)-propan-1-one(6.0 g, 22.1 mmol) in anhydrous dichloromethane (24 mL) was prepared andtert-butyldimethylsilyl trifluoromethanesulfonate (5.0 mL, 22.1 mmol)was added, followed by anhydrous triethylamine (3.54 mL, 25.4 mmol). Thereaction mixture was stirred at RT under a nitrogen atmosphere for 15 h.This gave a dark solution that was evaporated to give an oil. The oilwas dissolved in anhydrous dichloromethane (22 mL) and the solution wasadded dropwise to a solution of crotonaldehyde (3.66 mL, 44.2 mmol) andtitanium tetrachloride (1 M in dichloromethane, 44.2 mL, 44.2 mmol) indichloromethane (22 mL) at −78° C. under a nitrogen atmosphere. Thereaction mixture was stirred at −78° C. for 1 hour, before addition ofammonium chloride solution (30 mL). The stirred mixture was allowed towarm to RT before separating the layers. The aqueous layer was extractedwith dichloromethane (2×25 mL). The organic layers were combined, driedover sodium sulfate, filtered and evaporated to give a brown oil. Theoil was purified by silica gel chromatography using iso-hexanes/ethylacetate 4:1 to yield the title compound (6.7 g, 89%) as a colorlesssolid.

A solution of 5g (4.15 g, 12.1 mmol) in anhydrous dichloromethane (80mL) was prepared and 1,8-bis(dimethylamino)naphthalene (7.78 g, 36.3mmol) was added followed by trimethyloxonium tetrafluoroborate (3.6 g,24.2 mmol). The reaction mixture was stirred at RT for 3 h. The reactionmixture was treated with methanol (3 mL) and stirred for 5 min beforeadding hydrochloric acid (2 M, 200 mL) and ethyl acetate (250 mL). Themixture was filtered to remove an insoluble solid and the layers wereseparated. The aqueous layer was extracted with ethyl acetate (2×100mL). The organic layers were combined and washed with brine, dried oversodium sulfate, filtered and evaporated to give the title compound (4.80g, 100%) as a pale brown solid.

A solution of lithium hydroxide in water (2 M, 50 mL, 100 mmol) wasadded to a stirred solution of 5h (4.80 g, 12.1 mmol) in tetrahydrofuran(130 mL). The reaction mixture was heated to 60° C. for 15 h. Thereaction mixture was cooled to RT, before partially evaporating andadding hydrochloric acid (2 M, 150 mL). The mixture was extracted withethyl acetate (3×50 mL). The extract was dried over sodium sulfate,filtered and evaporated to give a brown oil (3.5 g). The oil waspurified by silica gel chromatography using iso-hexanes/diethyl ether1:1 to give the title compound (1.132 g, 59%) as a colorless oil.

A cooled (0° C.) solution of(S)-1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (2.174 g, 4.088 mmol) in anhydrousdichloromethane (50 mL) was treated with trimethylsilyltrifluoromethanesulfonate (1.2 mL, 6.814 mmol). After 1 h at 0° C., thereaction mixture was treated with N,N-diisopropylethylamine (2.4 mL,13.628 mmol) and the volatiles were removed in vacuo to afford thecorresponding amine as a yellow foam. To this amine was added 5i (539.0mg, 3.407 mmol), N,N-diisopropylethylamine (2.4 mL, 13.628 mmol) andacetonitrile (50 mL). The reaction mixture was cooled to 0° C. andtreated with 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (1.814 g, 4.770 mmol). After overnightstirring at RT the reaction was quenched with 1 M hydrochloric acid (100mL). The aqueous layer was extracted with ethyl acetate (2×). Theorganics were combined, dried over sodium sulfate, filtered and thevolatiles were removed in vacuo. The residue was purified by silica gelchromatography using a 50 g Isolute cartridge eluted with a continuousgradient of iso-hexanes/ethyl acetate 1:0 to 1:4 to afford the titlecompound (2.193 g, 93%) as a light yellow solid.

A solution of 5j (763.4 mg, 1.335 mmol) in tetrahydrofuran (25 mL) wassubsequently treated with zinc powder (1.920 g, 29.365 mmol) and asolution of ammonium acetate (1.543 g, 20.025 mmol) in water (5 mL).After overnight stirring the reaction was filtered through Celite andquenched with 2 M hydrochloric acid. The aqueous layer was extractedwith ethyl acetate (2×). The organics were combined, dried over sodiumsulfate, filtered and the volatiles were removed in vacuo. Residualacetic acid was azeotroped off with toluene to provide the titlecompound (566.4 mg, 96%) as a light orange solid.

A solution of 5k (246 mg, 0.6 mmol),(R)-1-(3-vinyl-isoquinolin-6-yl)-ethylamine dihydrochloride (162 mg, 0.6mmol) and N,N-diisopropylethylamine (387 mg, 0.52 mL, 3 mmol) inacetonitrile (20 mL) was stirred at RT under nitrogen.2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (320 mg, 0.84 mmol) was added and thereaction mixture was stirred at RT for 4 h. The solvent was evaporated.The residue was diluted with ethyl acetate and saturated sodium hydrogencarbonate solution. A small amount of methanol (5 mL) was added to thesuspension to give two clear layers. The organic layer was separated,washed with water and brine, dried over anhydrous sodium sulfate,filtered and evaporated. The residue was purified by silica gelchromatography using methanol/dichloromethane 1:20. The residue wastriturated with ether and the resulting solid was collected, washed withether and dried to afford the title compound (238 mg, 64%) as a palebrown solid.

A solution of 5l (91 mg, 0.15 mmol) in toluene (45 mL) was stirred at RTunder nitrogen. Hoveyda-Grubbs 2^(nd) generation catalyst (10 mg, 0.015mmol) was added and the reaction mixture was heated at reflux undernitrogen for 2 h. Additional Hoveyda-Grubbs 2^(nd) generation catalyst(10 mg, 0.015 mmol) was added and the reaction mixture was heated atreflux under nitrogen for 6 h. The reaction mixture was cooled to RT,silica gel was added and the reaction mixture was evaporated. Theresidue was purified by silica gel chromatography using a gradient ofethyl acetate/acetone 20:1 to 5:2. The residue was triturated with etherand the resulting solid was collected, washed with ether/iso-hexanes(1:1) and dried to afford the title compound (23 mg, 26%) as a whitesolid. ¹H NMR (300 MHz, CDCl₃) δ0.99 (d, J=6.7 Hz, 3H), 1.04 (d, J=6.7Hz, 3H), 1.44 (d, J=7.4 Hz, 3H), 1.53 (d, J=6.9 Hz, 3H), 1.61 (d, J=6.9Hz, 3H), 1.68-2.20 (m, 5H), 2.62-2.74 (m, 2H), 3.35-3.43 (m, 1H), 3.44(s, 3H), 3.72 (d, J=12.5 Hz, 1H), 3.88-3.92 (m, 2H), 4.23 (dd, J=8.9,6.0 Hz, 1H), 4.55-4.60 (m, 1H), 5.30-5.37 (m, 1H), 5.80-5.90 (m, 1H),6.38 (d, J=8.9 Hz, 1H), 6.43 (d, J=7.6 Hz, 1H), 6.93 (d, J=16.3 Hz, 1H),7.24 (dd, J=16.3, 8.7 Hz, 1H), 7.45 (d, J=8.7 Hz, 1H), 7.56 (d, J=8.5Hz, 1H), 7.69 (s, 1H), 7.93 (d, J=8.5 Hz, 1H), 9.17 (s, 1H). LCMS (m/z)579.3 [M+H], Tr=1.40 min.

Example 6

A solution of Compound 5 (11 mg, 0.019 mmol) in ethyl acetate (10 mL)containing 10% palladium on carbon (10 mg) was hydrogenated at RT andpressure for 3 h. The reaction mixture was filtered through filter aidand the filter pad was washed with ethyl acetate. The filtrate wasevaporated and the residue was purified by silica gel chromatographyusing a gradient of ethyl acetate/acetone 10:1 to 2:1. The residue wastriturated with ethyl acetate/ether (1:5) to afford the title compound(2.0 mg, 18%) as a white solid. ¹H NMR (300 MHz, CDCl₃) 0.93 (d, J=6.7Hz, 3H), 0.98 (d, J=6.7 Hz, 3H), 1.39 (d, J=6.9 Hz, 3H), 1.55-2.70 (m,8H), 1.95-2.50 (m, 5H), 2.64-2.73 (m, 2H), 2.95-3.08 (m, 2H), 3.35-3.42(m, 2H), 3.53 (s, 3H), 3.61 (d, J=12.1 Hz, 1H), 4.06-4.11 (m, 1H),4.50-4.57 (m, 1H), 5.25-5.32 (m, 1H), 5.64-5.70 (m, 1H), 6.39 (d, J=8.3Hz, 1H), 6.47-6.51 (m, 1H), 7.21-7.28 (m, 1H), 7.41 (d, J=8.8 Hz, 1H),7.62 (s, 1H), 7.69 (s, 1H), 7.92 (d, J=8.8 Hz, 1H), 9.15 (s, 1H). LCMS(m/z) 581.3 [M+H], Tr=1.38 min.

Example 7

A mixture of 7-bromo-2-chloro-quinoxaline (1.46 g, 6.00 mmol) andtributyl(1-ethoxyvinyl)tin (2.71 g, 2.54 mL, 7.50 mmol) in toluene (21mL) was degassed for 20 min. Bis(triphenylphosphine)palladium(II)dichloride (427 mg, 0.60 mmol) was added and the reaction mixturestirred under nitrogen and heated at 80° C. for 19 h before allowing tocool. The volatiles were evaporated and the residue suspended in1,4-dioxane (15 mL), 2 M aqueous hydrochloric acid (15 mL) was added andthe reaction mixture stirred for 45 min and then evaporated to removethe volatiles. The residue was diluted with water and extracted withethyl acetate (2×) and the combined organic extracts were washed withbrine, dried over magnesium sulfate, filtered and evaporated. Theproduct was purified by chromatography using silica gel doped with 10%w/w potassium carbonate eluting using a gradient of iso-hexanes/ethylacetate 1:0 to 9:1 to afford the title compound (836 mg, 56%) as ayellow solid.

Dichloro (p-cymene) ruthenium(II) dimer (12 mg, 0.019 mmol) and(1R,2R)-(−)-N-p-tosyl-1,2-diphenylethylenediamine (17 mg, 0.045 mmol)were suspended in degassed water (7.5 mL) and the mixture was degassedwith nitrogen for 15 min. The mixture was stirred at 70° C. undernitrogen for 90 min. The resulting turbid orange mixture was allowed tocool to RT. Solid 7a (948 mg, 3.78 mmol) followed by degassedtetrahydrofuran (7.5 mL) and sodium formate (1.29 g, 18.9 mmol) wereadded and the reaction mixture was degassed with nitrogen for 5 min. Thereaction mixture was vigorously stirred at 40° C. for 3 h and allowed tocool. It was then diluted with ethyl acetate and the mixture washed withwater (2×). The aqueous washes were back-extracted with ethyl acetateand the combined organics washed with brine, dried over magnesiumsulfate, filtered and evaporated. The residue was purified by silica gelchromatography eluting with iso-hexanes/ethyl acetate 2:1 to afford thetitle compound (814 mg, 85%) as a purple solid.

To a mixture of 7b (490 mg, 1.94 mmol), N,N-dicyclohexylmethylamine (416mg, 457 μL, 2.13 mmol) and tert-butyl 3-butenoate (648 mg, 739 μL, 4.56mmol) in toluene (19 mL) was addedbis(tri-tert-butylphosphine)palladium(0) (41 mg, 0.080 mmol) undernitrogen and the reaction mixture stirred and heated under reflux for 5h then allowed to cool. The mixture was evaporated and then purified bysilica gel chromatography using a gradient of iso-hexanes/ethyl acetate4:1 to 3:2 to afford the title compound (367 mg, 60%) as a yellow oil.

A solution of 1e (804 mg, 1.51 mmol) in tetrahydrofuran (37.7 mL) wasprepared and zinc powder (2.18 g, 33.3 mmol) was added followed by asolution of ammonium acetate (1.75 g, 22.7 mmol) in water (9.4 mL). Thereaction mixture was stirred at RT for 72 h. The reaction was filteredthrough hyflo-supercel washing through with ethyl acetate and saturatedaqueous potassium hydrogen sulfate. The mixture was treated with 1 Mhydrochloric acid (3 mL) and the layers were separated. The aqueouslayer was extracted with ethyl acetate (2×100 mL). The organic layerswere combined, washed with brine, filtered and evaporated to give acolorless gum. The residue was azeotroped with toluene (3×200 mL) togive the title compound (605 mg, quantitative yield) as a white solid.

To a stirred solution of 7d (456 mg, 1.14 mmol) and(E)-4-[3-((R)-1-hydroxy-ethyl)-quinoxalin-6-yl]-but-3-enoic acidtert-butyl ester (358 mg, 1.14 mmol) in dichloromethane (22 mL) wasadded 1-hydroxybenzotriazole containing approx. 20% water (270 mg, 1.60mmol) followed by 4-dimethylaminopyridine (139 mg, 1.14 mmol) andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (306 mg,1.60 mmol). The reaction was stirred for 18 h and then diluted withdichloromethane, washed with saturated ammonium chloride solution (2×),dried over sodium sulfate, filtered and evaporated. The residue waspurified by silica gel chromatography using a gradient ofiso-hexanes/ethyl acetate 3:1 to 0:1 to afford the title compound (335mg, 45%) as a white foam.

To a stirred solution of 7e (309 mg, 0.444 mmol) in dichloromethane (4.5mL) at 0° C. under nitrogen was added trimethylsilyltrifluoromethanesulfonate (346 mg, 359 μL, 1.56 mmol) and the reactionmixture was allowed to warm to RT over 2.5 h. N,N-diisopropylethylamine(164 mg, 221 μL, 1.27 mmol) was added and the reaction mixture stirredfor a further 10 min, evaporated and then suspended in acetonitrile (45mL). The stirred mixture was cooled to 0° C. and2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (236 mg, 0.622 mmol) andN,N-diisopropylethylamine (229 mg, 309 μL, 1.77 mmol) added. After 90min the reaction was quenched with a saturated ammonium chloridesolution and the mixture evaporated to remove organic volatiles. Theresidue was diluted with dichloromethane and the organic layer separatedand washed with saturated sodium bicarbonate (2×) and brine then driedover sodium sulfate, filtered and evaporated. The residue was purifiedby silica gel chromatography eluting with a gradient of ethylacetate/acetone 1:0 to 9:1. The residue was further purified by reversephase preparative HPLC using acetonitrile/water 3:7 to afford the titlecompound (7.6 mg, 3% over 2 steps) as a white solid. ¹H NMR (300 MHz,CD₃OD) δ1.00 (d, J=6.7 Hz, 3H), 1.01 (d, J=6.7 Hz, 3H), 1.62 (d, J=7.1Hz, 3H), 1.66-1.75 (m, 2H), 1.77 (d, J=6.9 Hz, 3H), 1.91-2.05 (m, 3H),2.72-2.82 (m, 1H), 2.98-3.08 (m, 1H), 3.38-3.41 (m, 1H), 3.78-3.84 (m,1H), 4.25 (d, J=10.5 Hz, 1H), 4.41 (br d, J=11.3 Hz, 1H), 5.68 (q, J=7.1Hz, 1H), 6.09 (q, J=6.9 Hz, 1H), 6.47 (d, J=16.3 Hz, 1H), 6.55-6.63 (m,1H), 7.68 (s, 1H), 7.99 (s, 2H), 8.84 (s, 1H). LCMS (m/z) 523.2 [M+H],Tr=1.75 min.

Examples 8 and 9

Dichloro (p-cymene) ruthenium(II) dimer (3 mg, 0.005 mmol) and(1R,2R)-(−)-N-p-tosyl-1,2-diphenylethylenediamine (4.4 mg, 0.012 mmol)were suspended in degassed water (2 mL) and the mixture was degassedwith nitrogen for 15 min. The mixture was stirred at 70° C. undernitrogen for 90 min. The resulting yellow solution was cooled to RT.1-(3-Chloro-isoquinolin-6-yl)-ethanone (206 mg, 1 mmol), sodium formate(340 mg, 5 mmol) and degassed tetrahydrofuran (1 mL) were added and thereaction mixture was degassed with nitrogen for 5 min. The reactionmixture was vigorously stirred at 40° C. for 2.5 h. The reaction mixturewas cooled to RT and was extracted with ethyl acetate. The organic layerwas separated, washed with water and brine, dried over sodium sulfate,filtered and evaporated. The residue was purified by silica gelchromatography using a gradient of iso-hexanes/ethyl acetate 4:1 to 2:1to afford the title compound (193 mg, 92%) as a white solid.

1,4-Dioxane (5 mL) was degassed with nitrogen, 8a (208 mg, 1 mmol),tributyl(vinyl)tin (951 mg, 0.9 mL, 3 mmol) andbis(triphenylphosphine)palladium(II) dichloride (70 mg, 0.1 mmol) wereadded and the reaction mixture was heated at 150° C. in a microwavereactor for 1 h. Additional tributyl(vinyl)tin (0.3 mL, 1 mmol) andbis(triphenylphosphine)palladium(II) dichloride (70 mg, 0.1 mmol) wereadded and the reaction mixture was heated at 150° C. in a microwavereactor for 1 h. The reaction mixture was cooled to RT and the mixturewas filtered through filter aid and the filter pad was washed with ethylacetate. The filtrate was evaporated and the residue was purified bysilica gel chromatography using a gradient of iso-hexanes/ethyl acetate4:1 to 2:1 followed by silica gel chromatography using iso-hexanes/ethylacetate 3:1 to afford the title compound (100 mg, 50%) as a white solid.

A solution of 1e (10.6 g, 20 mmol) in dichloromethane (300 mL) wasstirred at 0° C. under nitrogen. Trimethylsilyltrifluoromethanesulfonate (6.66 g, 5.4 mL, 30 mmol) was added dropwiseand the reaction mixture was stirred at 0° C. for 1 h. Cold saturatedsodium hydrogen carbonate solution (200 mL) was added and the reactionmixture was stirred at 0° C. for 15 min. The organic layer wasseparated, washed with saturated sodium hydrogen carbonate solution andbrine, dried over sodium sulfate, filtered and evaporated to afford(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (20 mmol), which was used crude in thenext step. A solution of(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (20 mmol) in acetonitrile (240 mL) wasstirred at 0° C. under nitrogen. (E)-Pent-3-enoic acid (2.20 g, 2.2 mL,22 mmol) and 1-hydroxybenzotriazole hydrate (3.82 g, 20 mmol, wettedwith not less than 20 wt. % water) andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (5.38 g, 28mmol) was added and the reaction mixture was stirred at 0° C. for 15 minand then at RT for 20 h. The solvent was evaporated and the residue waspartitioned between ethyl acetate and water. The organic extracts werecombined, washed with water and brine, dried over anhydrous sodiumsulfate, filtered and evaporated. The residue was purified by silica gelchromatography using iso-hexanes/ethyl acetate 3:1 to neat ethyl acetateto afford the title compound (9.0 g, 88%) as a white solid.

A solution of 8c (9.0 g, 17.5 mmol) in tetrahydrofuran (300 mL) wasstirred at RT under nitrogen. Zinc powder (25.0 g, 385 mmol) was addedfollowed by a solution of ammonium acetate (20.2 g, 263 mmol) in water(200 mL). The reaction mixture was stirred at RT under nitrogen for 18h. The reaction mixture was filtered through Celite and the filter padwas washed with water (100 mL) and ethyl acetate (200 mL). The organiclayer was separated and the solvent was evaporated to ca. 100 mL and thesolution was extracted with water (100 mL). The aqueous layers werecombined, saturated ammonium chloride solution (150 mL) was added andthe solution was acidified to pH 1 with 2 M aqueous hydrochloric acid.The solution was extracted with ethyl acetate and the organic extractswere combined, washed with brine, dried over anhydrous magnesiumsulfate, filtered and evaporated. The residue was co-evaporated withtoluene (3×) to afford(S)-1-{(S)-2-[(S)-3-methyl-2-((E)-pent-3-enoylamino)-butyrylamino]-propionyl}-hexahydro-pyridazine-3-carboxylicacid (5.7 g, 85%) as a white solid which was used in the next reaction.A solution of(S)-1-{(S)-2-[(S)-3-methyl-2-((E)-pent-3-enoylamino)-butyrylamino]-propionyl}-hexahydro-pyridazine-3-carboxylicacid (5.16 g, 13.5 mmol) in dichloromethane (280 mL) and tetrahydrofuran(20 mL) was stirred at RT under nitrogen.(R)-1-(3-Vinyl-isoquinolin-6-yl)-ethanol (2.69 g, 13.5 mmol) was addedfollowed by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride(3.63 g, 18.9 mmol) and 4-(dimethylamino)pyridine (1.64 g, 13.5 mmol)and the reaction mixture was stirred at RT for 6 h. Dichloromethane (200mL) was added and the solution was washed with aqueous citric acidsolution (pH 2-3). The organic layer was separated, washed with waterand brine, dried over anhydrous sodium sulfate, filtered and evaporated.The residue was purified by silica gel chromatography using a gradientof iso-hexanes/ethyl acetate 3:1 to neat ethyl acetate followed bysilica gel chromatography using iso-hexanes/ethyl acetate 1:8 to affordthe title compound (3.91 g, 51%) as a white solid.

A solution of 8d (1.13 g, 2 mmol) in toluene (600 mL) was stirred at RTunder nitrogen for 15 min. Hoveyda-Grubbs 2^(nd) generation catalyst(125 mg, 0.2 mmol) was added and the reaction mixture was heated atreflux under nitrogen for 30 min. Additional Hoveyda-Grubbs 2^(nd)generation catalyst (125 mg, 0.2 mmol) was added and the reactionmixture was heated at reflux for 30 min. The reaction mixture was cooledto RT. The majority of the solvent was evaporated, silica gel was addedand the solvent was evaporated. The residue was purified by silica gelchromatography using a gradient of ethyl acetate/acetone 9:1 to 3:2followed by silica gel chromatography using ethyl acetate/methanol 40:1.The residue was triturated with ethyl acetate/ether (1:4) and theresulting solid was collected, washed with ethyl acetate/ether (1:4) anddried to afford a −10:1 mixture of Compound 9 and Compound 8, as a palebrown solid (245 mg). A sample of the mixture was purified by reversephase preparative HPLC to afford Compound 8 (4 mg) as a white solid. ¹HNMR (300 MHz, CD₃OD) δ1.01 (d, J=6.5 Hz, 3H), 1.02 (d, J=6.5 Hz, 3H),1.37 (d, J=7.1 Hz, 3H), 1.65 (d, J=6.5 Hz, 3H), 1.70-2.15 (m, 5H),2.95-3.05 (m, 1H), 3.70-3.76 (m, 1H), 4.03-4.10 (m, 2H), 4.12 (d, J=8.3Hz, 1H), 5.64 (q, J=7.1 Hz, 1H), 6.10-6.20 (m, 2H), 6.98 (d, J=11.3 Hz,1H), 7.65 (d, J=8.5 Hz, 1H), 7.93 (s, 1H), 8.05-8.10 (m, 2H), 9.21 (s,1H). LCMS (m/z) 522.3 [M+H], Tr=1.25 min.

Compound 9, ¹H NMR (300 MHz, CDCl₃) δ0.97 (d, J=6.9 Hz, 3H), 0.99 (d,J=6.7 Hz, 3H), 1.53 (d, J=6.9 Hz, 3H), 1.71 (d, J=6.9 Hz, 3H), 1.75-2.70(m, 6H), 3.20-3.40 (m, 2H), 3.63-3.77 (m, 2H), 4.23-4.29 (m, 1H),4.53-5.57 (m, 1H), 5.65-5.76 (m, 1H), 6.04 (q, J=6.7 Hz, 1H), 6.38-6.53(m, 3H), 6.72 (d, J=16.3 Hz, 1H), 7.39 (d, J=8.3 Hz, 1H), 7.55 (s, 1H),7.72 (s, 1H), 7.92 (d, J=8.3 Hz, 1H), 9.13 (s, 1H). LCMS (m/z) 522.0[M+H], Tr=1.40 min.

Example 10

10a was prepared in the same manner as 4d using 3-butenoic acid insteadof 4,4-dimethyl-hept-6-enoic acid in 66% yield.

10b was prepared in the same manner as 4e using(S)-1-[(S)-2-((S)-2-but-3-enoylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester instead of(S)-1-{(S)-2-[(S)-2-(4,4-dimethyl-hept-6-enoyloxy)-3-methyl-butyrylamino]-propionyl}-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester in 84% yield.

10c was prepared in the same manner as 8d using(S)-1-[(S)-2-((S)-2-but-3-enoylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid instead of(S)-1-{(S)-2-[(S)-3-methyl-2-((E)-pent-3-enoylamino)butyrylamino]-propionyl}-hexahydro-pyridazine-3-carboxylicacid in 84% yield.

Compound 9 was prepared in the same manner as Compound 5 using(S)-1-[(S)-2-((S)-2-but-3-enoylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (R)-1-(3-vinyl-isoquinolin-6-yl)-ethyl ester instead of(S)-1-{(S)-2-[(S)-2-((E)-(2R,3R)-3-methoxy-2-methyl-hex-4-enoylamino)-3-methyl-butyrylamino]-propionyl}-hexahydro-pyridazine-3-carboxylicacid [(R)-1-(3-vinyl-isoquinolin-6-yl)-ethyl]-amide in 9% yield.

A solution of Compound 9 (16 mg, 0.03 mmol) in ethyl acetate (5 mL) andtetrahydrofuran (2 mL) containing 10% palladium on carbon (15 mg) washydrogenated at RT and pressure for 3 h. The reaction mixture wasfiltered through filter aid and the filter pad was washed with ethylacetate. The filtrate was evaporated and the residue was triturated withether (2×2 mL) and the resulting solid was dried to afford the titlecompound (7.6 mg, 48%) as an off-white solid. ¹H NMR (300 MHz, CDCl₃)δ0.90 (d, J=6.7 Hz, 3H), 0.95 (d, J=6.5 Hz, 3H), 1.50 (d, J=7.1 Hz, 3H),1.68 (d, J=6.5 Hz, 3H), 1.60-2.40 (m, 9H), 2.70-3.10 (m, 3H), 3.68-3.75(m, 1H), 3.90 (d, J=11.8 Hz, 1H), 4.32-4.37 (m, 1H), 4.44-4.49 (m, 1H),5.83-5.92 (m, 1H), 6.05-6.11 (m, 2H), 6.33 (d, J=7.8 Hz, 1H), 7.34 (s,1H), 7.36 (d, J=8.3 Hz, 1H), 7.80 (s, 1H), 7.93 (d, J=8.3 Hz, 1H), 9.17(s, 1H). LCMS (m/z) 524.3 [M+H], Tr=0.59 min.

Examples 11 and 12

To a stirred solution of 1e (10.6 g, 20.0 mmol) in dichloromethane (400mL), at 0° C. under nitrogen, was added trimethylsilyltrifluoromethanesulfonate (6.67 g, 5.43 mL, 30.0 mmol) and the reactionmixture stirred at 0° C. for 2 h. N,N-Diisopropylethylamine (10.3 g,13.9 mL, 80.0 mmol) was added and the mixture allowed to warm to ambienttemperature. The volatiles were evaporated and the residue suspended inacetonitrile (250 mL). The stirred mixture was cooled to 0° C. under ablanket of nitrogen and then N,N-diisopropylethylamine (10.3 g, 13.9 mL,80 mmol) and 3-butenoic acid (1.89 g, 1.86 mL, 4.40 mmol) added,followed by 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (10.6 g, 28.0 mmol), portionwise. Thereaction mixture was allowed to warm to ambient temperature and stirredfor 18 h before evaporating. The residue was diluted with ethyl acetateand then washed successively with saturated sodium bicarbonate solution,water, 2 M hydrochloric acid, water then brine, dried over magnesiumsulfate, filtered and evaporated. The residue was purified by silica gelchromatography using a gradient of iso-hexanes/ethyl acetate 1:1 to 0:1to afford a ˜1:1 mixture of(S)-1-[(S)-2-((S)-2-but-3-enoylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester and(S)-1-{(S)-2-[(S)-2-((E)-but-2-enoylamino)-3-methyl-butyrylamino]-propionyl}-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester as an orange foam (7.23 g, 72%).

To a stirred solution of a mixture of(S)-1-[(S)-2-((S)-2-but-3-enoylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester and(S)-1-{(S)-2-[(S)-2-((E)-but-2-enoylamino)-3-methyl-butyrylamino]-propionyl}-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (˜1:1, 0.99 g, 2.00 mmol) intetrahydrofuran (40 mL) was added zinc powder (2.86 g, 44.0 mmol) and asolution of ammonium acetate (2.31 g, 30.0 mmol) in water (25 mL). Thereaction mixture was allowed to warm to ambient temperature and stirredfor 18 h, then diluted with ethyl acetate and the mixture filtered. Fromthe filtrate the aqueous layer was separated and diluted with an equalamount of saturated ammonium chloride solution and then acidified to pH1 with 2 M hydrochloric acid. The aqueous layer was extracted with ethylacetate (2×) and the combined organic layers washed with brine, driedover magnesium sulfate, filtered and evaporated. The residue wasazeotroped with toluene (3×) to give a mixture of the title compounds(˜1:1, 466 mg, 63%) as a yellow foam.

A mixture of 11a and 12b (˜1:1, 250 mg, 0.68 mmol) in acetonitrile (20mL) was stirred at RT under nitrogen.(R)-1-(3-vinyl-isoquinolin-6-yl)-ethylamine dihydrochloride (136 mg, 0.5mmol) and N,N-diisopropylethylamine (323 mg, 0.44 mL, 2.5 mmol) wasadded followed by 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methanaminium (266 mg, 0.7 mmol) and thereaction mixture was stirred at RT for 18 h. The solvent was evaporated.The residue was diluted with ethyl acetate and the solution was washedwith saturated sodium hydrogen carbonate solution, water and brine. Theorganic extract was dried over anhydrous magnesium sulfate, filtered andevaporated. The residue was purified by silica gel chromatography usinga gradient of ethyl acetate to methanol/ethyl acetate 1:5 to afford a˜1:1 mixture of(S)-1-[(S)-2-((S)-2-but-3-enoylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid [(R)-1-(3-vinyl-isoquinolin-6-yl)-ethyl]-amide and(S)-1-{(S)-2-[(S)-2-((E)-but-2-enoylamino)-3-methyl-butyrylamino]-propionyl}-hexahydro-pyridazine-3-carboxylicacid [(R)-1-(3-vinyl-isoquinolin-6-yl)-ethyl]-amide as a white solid(153 mg, 56%) which was used in the next step without furtherpurification.

A solution of a mixture of(S)-1-[(S)-2-((S)-2-but-3-enoylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid [(R)-1-(3-vinyl-isoquinolin-6-yl)-ethyl]-amide and(S)-1-{(S)-2-[(S)-2-((E)-but-2-enoylamino)-3-methyl-butyrylamino]-propionyl}-hexahydro-pyridazine-3-carboxylicacid [(R)-1-(3-vinyl-isoquinolin-6-yl)-ethyl]-amide (˜1:1, 150 mg, 0.27mmol) in toluene (70 mL) was stirred at RT under nitrogen.Hoveyda-Grubbs 2^(nd) generation catalyst (17 mg, 0.027 mmol) was addedand the reaction mixture was heated at reflux under nitrogen for 90 min.Additional Hoveyda-Grubbs 2^(nd) generation catalyst (17 mg, 0.027 mmol)was added and the reaction mixture was heated at reflux under nitrogenfor 90 min. The reaction mixture was cooled to RT, silica gel was addedand the solvent was evaporated. The residue was purified by silica gelchromatography using a gradient of ethyl acetate to ethylacetate/acetone 1:1. The residue was further purified by preparativethin layer chromatography using methanol/ethyl acetate 1:4 followed bysilica gel chromatography using dichloromethane/methanol 20:1 to givethe title compounds. Compound 11 (1.3 mg, 1%) as a white solid: ¹H NMR(300 MHz, CD₃OD) δ1.01 (d, J=6.7 Hz, 3H), 1.02 (d, J=6.7 Hz, 3H), 1.61(d, J=7.1 Hz, 3H), 1.66 (d, J=7.1 Hz, 3H), 1.80-2.05 (m, 5H), 2.71-2.82(m, 1H), 2.99-3.05 (m, 1H), 3.32-3.43 (m, 1H), 3.64-3.75 (m, 1H), 4.27(d, J=10.1 Hz, 1H), 4.40-4.53 (m, 2H), 5.12 (q, J=7.1 Hz, 1H), 5.62 (q,J=7.1 Hz, 1H), 6.47-6.56 (m, 1H), 6.62 (d, J=15.8 Hz, 1H), 7.43 (s, 1H),7.61 (dd, J=8.5, 1.6 Hz 1H), 7.74 (s, 1H), 8.05 (d, J=8.5 Hz, 1H), 9.13(s, 1H). LCMS (m/z) 521.3 [M+H], Tr=0.97 min.

Compound 12 (1.7 mg, 1.2%) as a white solid: ¹H NMR (300 MHz, CD₃OD)δ0.98 (d, J=6.7 Hz, 3H), 1.03 (d, J=6.7 Hz, 3H), 1.47 (d, J=7.1 Hz, 3H),1.60 (d, J=6.9 Hz, 3H), 1.65-2.16 (m, 5H), 2.81-2.89 (m, 1H), 3.61-3.69(m, 1H), 4.04 (d, J=6.9 Hz, 1H), 4.30-4.36 (m, 1H), 4.75 (d, J=11.6 Hz,1H), 5.23 (q, J=7.1 Hz, 1H), 5.90 (q, J=6.9 Hz, 1H), 6.36 (d, J=13.1 Hz,1H), 6.92 (d, J=13.1 Hz, 1H), 7.65 (d, J=8.5 Hz 1H), 7.88 (s, 1H), 8.05(d, J=8.5 Hz 1H), 8.46 (s, 1H), 9.16 (s, 1H). LCMS (m/z) 507.2 [M+H],Tr=0.95 min.

Example 13

A suspension of (S)-1-(7-bromo-quinolin-2-yl)-2,2,2-trifluoro-ethylaminehydrochloride (Asiba Pharmatech, Edison, N.J., USA, 397 mg, 1.16 mmol)in dichloromethane (10 mL) was cooled using an ice bath. Triethylamine(985 μL, 3.48 mmol) was added and the reaction was stirred until ahomogenous solution was observed. Di-tert-butyl dicarbonate (380 mg,1.74 mmol) in dichloromethane (5 mL) was then added and the reaction wasleft to stir overnight. Di-tert-butyl dicarbonate (127 mg, 0.58 mmol)was added and the reaction was stirred for a further 6 h. Di-tert-butyldicarbonate (253 mg, 1.16 mmol) was added along with 4 Å molecularsieves and the reaction was left to stir overnight. The reaction waswashed with water and brine and the organics were dried over anhydroussodium sulfate, filtered and concentrated. The residue was purified bysilica gel chromatography using a stepwise gradient from 1:0 to 7:3iso-hexanes/ethyl acetate to afford the title compound (343 mg, 73%) asan orange solid.

To a 3-necked flask, fitted with stirrer bar and thermometer, under anatmosphere of nitrogen and containing magnesium turnings (3.02 g, 0.124mol) was added anhydrous tetrahydrofuran (105 mL). A single crystal ofiodine was added to the reaction followed by 1-chloro-3-methyl-but-2-ene(2 mL, 17.75 mmol). The reaction was stirred for 20 min until all colorhad left the solution. 1-chloro-3-methyl-but-2-ene (5.57 mL, 49.45 mmol)was added. A gradual temperature increase was observed until thereaction had reached reflux. The reaction was left to stir for 1 hour,allowing the reaction to return to RT. The reaction was then transferredvia cannula to a flask containing solid carbon dioxide (50 g). Thereaction was then stirred for 2 h. A cooling ice bath was added and thereaction was quenched with 2 M hydrochloric acid. The reaction mixturewas then extracted with diethyl ether. The organics were then dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by silica gel chromatography using a stepwise gradient from 1:0to 9:1 iso-hexanes/ethyl acetate to afford the title compound (2.69 g,35%) as a pale yellow oil. ¹H NMR (300 MHz, CDCl₃) δ1.35 (s, 6H),5.10-5.21 (m, 2H), 6.07 (dd, J=17.6, 10.5 Hz, 1H).

13a (343 mg, 0.846 mmol) and 13b (106 mg, 0.931 mmol) were placed in amicrowave vessel and dissolved in acetonitrile (3 mL). Palladium(II)acetate (19 mg, 0.0846 mmol), tri(o-tolyl)phosphine (51 mg, 0.169 mmol)and triethylamine (236 μL, 1.69 mmol) were added and the vessel wassealed before being irradiated in the microwave for 30 min, using fixedhold time, on high absorption at 100° C. The solvent was removed and theresidue was taken up in a mixture of water and ethyl acetate. The phaseswere separated and the aqueous was extracted with ethyl acetate. Thecombined organics were then dried over anhydrous sodium sulfate,filtered and concentrated. The residue was purified by silica gelchromatography using a stepwise gradient of iso-hexanes/ethyl acetatefrom 1:0 to 6:4. The isolated material was subjected to a secondpurification using the same conditions to afford the title compound (142mg, 38%) as a pale yellow oil.

(S)-1-[(S)-2-((S)-2-Amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid ethyl ester (210 mg, 0.486 mmol) and 13c (142 mg, 0.324 mmol) weredissolved in N,N-dimethylformamide (5 mL), under an atmosphere ofnitrogen, and cooled using an ice bath.2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (203 mg, 0.535 mmol) andN,N-diisopropylethylamine (423 μL, 2.43 mmol) were then added and thereaction was allowed to slowly warm to RT and left to stir overnight.The reaction was diluted with water and extracted with ethyl acetate.The combined organics were dried over anhydrous sodium sulfate, filteredand concentrated. The residue was purified by silica gel chromatographyusing a stepwise gradient of iso-hexanes/ethyl acetate from 1:0 to 0:1to afford the product contaminated with residual solvent. Toluene wasadded and the solution was concentrated to afford the title compound (79mg, 29%) as an orange solid.

13d (79 mg, 0.0925 mmol) was dissolved in tetrahydrofuran (2 mL) andcooled using an ice bath. Methanol (1 mL) and water (1 mL) were thenadded followed by lithium hydroxide monohydrate (15 mg, 0.37 mmol). Thereaction was then left to stir for 1 hour. 1 M hydrochloric acid wasadded until the solution was pH 2. The solvent was removed and theresultant solid was sequentially azeotroped with methanol, thenacetonitrile and finally toluene to afford the title compound (67 mg,100%) as a yellow solid.

13e (66 mg, 0.0916 mmol) was dissolved in 4 M hydrochloric acid in1,4-dioxane (2 mL) and left to stir for 30 min. The solvent was removedand the resultant solid was triturated with diethyl ether and dried toafford the title compound (50 mg, 83%) as a pale yellow solid.

13f (50 mg, 0.0761 mmol) was dissolved in dichloromethane (76 mL), underan atmosphere of nitrogen and cooled using an ice bath.2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (58 mg, 0.152 mmol) andN,N-diisopropylethylamine (53 μL, 0.304 mmol) were then added and thereaction was allowed to slowly warm to RT and left to stir overnight.The solvent was removed and the residue was purified by silica gelchromatography using 7:3 iso-hexanes/acetone. The residue wasre-purified by silica gel chromatography using a stepwise gradient ofiso-hexanes/acetone from 1:0 to 1:1. The residue was then eluted throughan HPLC system fitted with a Phenomenex Gemini 10μ 110 A, 250×21.2 mmcolumn using a continuous gradient of acetonitrile/water from 1:4 to 1:0flow at 20 mL/min to afford the title compound (7 mg, 15%) as a solid.¹H NMR (300 MHz, CDCl₃) δ0.86-1.08 (m, 6H), 1.25-1.54 (m, 8H), 1.57-1.81(m, 5H), 1.88-2.00 (m, 1H), 2.37-2.80 (m, 1H), 3.32-3.65 (m, 1H),3.69-3.97 (m, 1H), 4.22-4.39 (m, 1H), 4.50-4.68 (m, 1H), 5.74-6.05 (m,2H), 6.22-6.39 (m, 1H), 6.44-6.54 (m, 1H), 6.63-6.76 (m, 1H), 7.45 (appt, J=8.5 Hz, 1H), 7.56 (app t, J=7.6 Hz, 1H), 7.81 (d, J=8.3 Hz, 1H),8.19 (s, 1H), 8.76-9.09 (m, 1H). LCMS (m/z) 603.1 [M+H], Tr=2.59 min.

Example 14

Dichloro (p-cymene) ruthenium(II) dimer (24 mg, 0.040 mmol) and(1R,2R)-(−)-N-p-tosyl-1,2-diphenylethylenediamine (35 mg, 0.096 mmol)were suspended in degassed water (16 mL) and the mixture was degassedwith nitrogen for 15 min. The mixture was stirred at 70° C. undernitrogen for 90 min. The resulting turbid orange mixture was allowed tocool to RT. Solid 1-(6-bromo-quinolin-3-yl)-ethanone (prepared as inWO2011/063233, 1.92 g, 7.68 mmol) followed by degassed tetrahydrofuran(16 mL) and sodium formate (2.72 g, 40 mmol) were added and the reactionmixture was degassed with nitrogen for 5 min, further degassedtetrahydrofuran (5 mL) was added and the mixture degassed for anotherminute. The reaction mixture was vigorously stirred at 40° C. for 21 hand allowed to cool. It was then diluted with ethyl acetate and themixture washed with water then brine, dried over magnesium sulfate,filtered and evaporated. The residue was purified by silica gelchromatography using a gradient of iso-hexanes/ethyl acetate 7:3 to 0:1to afford the title compound (1.65 g, 85%) as a brown solid.

To a mixture of 14a (356 mg, 1.41 mmol), N,N-dicyclohexylmethylamine(275 mg, 301 μL, 1.41 mmol) and tert-butyl 3-butenoate (470 mg, 537 μL,3.31 mmol) in toluene (14 mL) was addedbis(tri-tert-butylphosphine)palladium(0) (30 mg, 0.058 mmol) undernitrogen and the reaction mixture stirred and heated under reflux for 90min then allowed to cool. The mixture was evaporated and then purifiedby silica gel chromatography using a gradient of iso-hexanes/ethylacetate 4:1 to 2:3 to afford the title compound as a yellow oil (144 mg,33%).

To a stirred solution of(S)-1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (189 mg, 0.473 mmol) and 14b (148 mg, 0.473 mmol) indichloromethane (10 mL) was added 1-hydroxybenzotriazole containingapprox. 20% water (89 mg, 0.662 mmol) followed by4-dimethylaminopyridine (58 mg, 0.473 mmol) thenN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (127 mg,0.662 mmol). The reaction was stirred for 62 h and then diluted withdichloromethane, washed with saturated ammonium chloride solution (2×)and brine, dried over magnesium sulfate, filtered and evaporated.Purified by silica gel chromatography using a gradient ofiso-hexanes/ethyl acetate 3:1 to 2:3 afforded the title compound (126mg, 38%) as a white foam.

To a stirred solution of 14c (126 mg, 0.181 mmol) in dichloromethane(1.8 mL) at 0° C. under nitrogen was added trimethylsilyltrifluoromethanesulfonate (141 mg, 147 μL, 0.635 mmol) and the reactionmixture was allowed to warm to ambient temperature over 1.75 h. Furthertrimethylsilyl trifluoromethanesulfonate (20 mg, 21 μL, 0.091 mmol) wasadded and the reaction mixture was stirred for a further 15 min beforethe addition of N,N-diisopropylethylamine (164 mg, 221 μL, 1.27 mmol).After a further 10 min of stirring the reaction mixture was evaporatedand then suspended in acetonitrile (18.1 mL). The stirred mixture wascooled to 0° C. and then2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (96 mg, 0.253 mmol) andN,N-diisopropylethylamine (94 mg, 126 μL, 0.724 mmol) added. After 45min the reaction was quenched with saturated ammonium chloride solution(10 mL) and the mixture evaporated to remove organic volatiles. Theresidue was diluted with dichloromethane and the aqueous layer separatedand extracted with dichloromethane. The organic extracts were washedwith saturated sodium bicarbonate, water and then brine, dried oversodium sulfate, filtered and evaporated. The residue was purified bysilica gel chromatography eluting with ethyl acetate/acetone 9:1 then bypreparative reverse phase HPLC using a gradient of acetonitrile/water3:7 to 1:1 to afford the title compound (6.5 mg, 7% over 2 steps) as awhite solid. ¹H NMR (300 MHz, CD₃OD) δ1.00 (d, J=6.7 Hz, 3H), 1.03 (d,J=6.7 Hz, 3H), 1.61 (d, J=7.1 Hz, 3H), 1.75 (d, J=6.7 Hz, 3H), 1.91-2.01(m, 3H), 2.68-2.79 (m, 1H), 3.00 (dd, J=14.5, 3.6 Hz, 1H), 3.35-3.42 (m,1H), 3.73-3.82 (m, 1H), 4.29 (d, J=9.6 Hz, 1H), 4.41 (d, J=12.9 Hz, 1H),4.70 (d, J=12.3 Hz, 1H), 5.47 (q, J=7.3 Hz, 1H), 6.15 (q, J=6.6 Hz, 1H),6.30-6.40 (m, 1H), 6.63 (d, J=16.1 Hz, 1H), 7.55 (s, 1H), 7.82 (dd,J=8.7, 1.5 Hz, 1H), 7.95 (d, J=8.7 Hz, 1H), 8.21 (s, 1H), 8.79 (d, J=1.8Hz, 1H). LCMS (m/z) 522.2 [M+H], Tr=1.60 min.

Example 15

To a stirred mixture of 3-bromo-quinoline-6-carboxylic acid (prepared asin WO2011/090935, 1.94 g, 7.70 mmol) in tetrahydrofuran (77 mL) wasadded 2-chloro-4,6-dimethoxy-1,3,5-triazine (2.03 g, 11.5 mmol) andN-methylmorpholine (2.34 g, 2.54 mL, 23.1 mmol). The reaction mixturewas stirred for 90 min and then N,O-dimethylhydroxylamine hydrochloride(751 mg, 7.70 mmol) added in one portion. The reaction mixture wasstirred for a further 17 h and further N,O-dimethylhydroxylaminehydrochloride (375 mg, 3.85 mmol) was added and then after 5 h a furtherportion (175 mg, 1.80 mmol). The reaction mixture was stirred for afurther hour and then diluted with dichloromethane, washed with waterand then saturated ammonium chloride solution. The organic layer wasdried over sodium sulfate, filtered and evaporated. The residue waspurified by silica gel chromatography eluting with a gradient ofiso-hexanes/ethyl acetate 4:1 to 7:3 to afford the title compound (1.43g, 66%) as an off-white solid.

To a stirred solution of methyl magnesium chloride (3 M intetrahydrofuran, 4.85 mL, 14.5 mmol) in tetrahydrofuran (10 mL) undernitrogen was added a solution of 15a (1.43 g, 4.85 mmol) intetrahydrofuran (20 mL). The reaction mixture was stirred for 1 hour andthen quenched with saturated ammonium chloride solution. The mixture wasdiluted with diethyl ether and water, the organic layer separated andwashed with further water and the combined aqueous washes back-extractedwith diethyl ether. The combined organic extracts were dried overmagnesium sulfate, filtered and evaporated. The residue was purified bysilica gel chromatography eluting with iso-hexanes/ethyl acetate 4:1 toafford the title compound as a white solid which was taken directly intothe next step.

Dichloro (p-cymene) ruthenium(II) dimer (9 mg, 0.015 mmol) and(1R,2R)-(−)-N-p-tosyl-1,2-diphenylethylenediamine (14 mg, 0.037 mmol)were suspended in degassed water (9 mL) and the mixture was degassedwith nitrogen for 15 min. The mixture was stirred at 70° C. undernitrogen for 90 min. The resulting turbid orange mixture was allowed tocool to RT. Solid 1-(3-bromo-quinolin-6-yl)-ethanone (771 mg, 3.08 mmol)and sodium formate (2.72 g, 40 mmol) followed by degassedtetrahydrofuran (4.5 mL) were added and the reaction mixture wasdegassed with nitrogen for 5 min. The reaction mixture was vigorouslystirred at 40° C. for 3 h and allowed to cool. It was then diluted withethyl acetate and the mixture washed with water then brine, dried oversodium sulfate, filtered and evaporated. The residue was purified bysilica gel chromatography using a gradient of iso-hexanes/ethyl acetate3:2 to 1:1 to afford the title compound (616 mg, 79%) as a grey solid.

To a solution of 15c (560 mg, 2.22 mmol) in toluene (20 mL) and1,4-dioxane (5 mL) was added N,N-dicyclohexylmethylamine (740 μL, 3.46mmol) and tert-butyl 3-butenoate (741 mg, 844 μL, 5.22 mmol) was addedbis(tri-tert-butylphosphine)palladium(0) (68 mg, 0.13 mmol) undernitrogen and the reaction mixture stirred and heated under reflux for 6h then allowed to cool. The mixture was evaporated and then purified bysilica gel chromatography using a gradient of iso-hexanes/ethyl acetate4:1 to 0:1 to give a yellow gum. The gum was suspended in ethyl acetateand washed with saturated ammonium chloride solution (2×) followed bywater then brine, dried over sodium sulfate, filtered and evaporated toafford the title compound (300 mg, 43%) as a yellow oil.

To a stirred slurry of(S)-1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (325 mg, 0.812 mmol), 15d (231 mg, 0.738 mmol) and powdered 4 Åmolecular sieves in dichloromethane (16 mL) was addedN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (198 mg,1.03 mmol), 1-hydroxybenzotriazole hydrate containing approx. 20% water(174 mg, 1.03 mmol) followed by 4-dimethylaminopyridine (90 mg, 0.738mmol). The reaction was stirred under nitrogen for 16 h and thenfiltered and the solid washed with dichloromethane. The filtrate waswashed with saturated ammonium chloride solution (2×) and water, driedover sodium sulfate, filtered and evaporated. The residue was purifiedby silica gel chromatography using a gradient of iso-hexanes/ethylacetate 1:1 to 0:1 to afford the title compound (307 mg, 60%) as ayellow foam.

To a stirred solution of 15e (150 mg, 0.216 mmol) in dichloromethane (2mL) at 0° C. under nitrogen was added trimethylsilyltrifluoromethanesulfonate (168 mg, 174 μL, 0.755 mmol) dropwise and thereaction mixture was allowed to warm to ambient temperature over 2.25 h.N,N-Diisopropylethylamine (195 mg, 263 μL, 1.51 mmol) was added and thereaction mixture stirred for a further 10 min, evaporated and thensuspended in acetonitrile (21.6 mL). The stirred mixture was cooled to0° C. and then 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (115 mg, 0.320 mmol) andN,N-diisopropylethylamine (111 mg, 150 μL, 0.864 mmol) added. After 1 hthe reaction was quenched with saturated ammonium chloride solution andthe mixture evaporated to remove organic volatiles. The residue wasdiluted with dichloromethane and the organic layer separated and washedwith saturated ammonium chloride (2×) and brine then dried over sodiumsulfate, filtered and evaporated. The residue was purified bypreparative reverse phase HPLC using a gradient of acetonitrile/water3:7 to 11:9 to afford the title compound (4.6 mg, 4% over 2 steps) as anoff-white solid. ¹H NMR (300 MHz, CD₃OD) δ1.01 (app t, J=6.7 Hz, 6H),1.62 (d, J=7.1 Hz, 3H), 1.70 (d, J=6.7 Hz, 3H), 1.75-1.85 (m, 1H),1.91-2.05 (m, 3H), 2.70-2.80 (m, 1H), 2.98-3.06 (m, 1H), 3.36-3.45 (m,1H), 3.74-3.84 (m, 1H), 4.26 (d, J=10.5 Hz, 1H), 4.42 (br d, J=12.3 Hz,1H), 4.72-4.78 (m, 1H), 5.55 (q, J=7.1 Hz, 1H), 6.09 (q, J=6.5 Hz, 1H),6.38-6.41 (m, 1H), 6.71 (d, J=16.1 Hz, 1H), 7.68 (dd, J=8.9, 1.9 Hz,1H), 7.88-8.01 (m, 3H), 8.86 (d, J=2.0 Hz, 1H). LCMS (m/z) 522.3 [M+H],Tr=1.64 min.

Example 16

A mixture of 7-bromo-2-chloro-quinoline (1.05 g, 4.32 mmol) andtributyl(1-ethoxyvinyl)tin (1.95 g, 1.83 mL, 5.40 mmol) in toluene (21mL) was degassed for 20 min. Bis(triphenylphosphine)palladium(II)dichloride (302 mg, 0.432 mmol) was added and the reaction mixturestirred under nitrogen and heated at 80° C. for 24 h before allowing tocool. The volatiles were evaporated and the residue suspended in1,4-dioxane (10 mL) and 2 M aqueous hydrochloric acid (5 mL) was addedand the reaction mixture stirred for 30 min and then evaporated toremove organics. The residue was diluted with ethyl acetate and waterand the organic layer washed with brine, dried over sodium sulfate,filtered and evaporated. The product was purified on silica gel dopedwith 10% w/w potassium carbonate eluting using a gradient ofiso-hexanes/ethyl acetate 9:1 to 4:1 to afford the title compound (422mg, 48%) as a yellow solid.

Dichloro(p-cymene)ruthenium(II) dimer (8.5 mg, 0.014 mmol) and(1R,2R)-(−)-N-p-tosyl-1,2-diphenylethylenediamine (12.1 mg, 0.033 mmol)were suspended in degassed water (5.5 mL) and the mixture was degassedwith nitrogen for 20 min. The mixture was stirred at 70° C. undernitrogen for 90 min. The resulting turbid orange mixture was allowed tocool to RT. Solid 16a (571 mg, 2.78 mmol) and sodium formate (945 mg,13.9 mmol) followed by degassed tetrahydrofuran (5.5 mL) were added andthe reaction mixture was degassed with nitrogen for 5 min. The reactionmixture was vigorously stirred at 40° C. for 4 h and allowed to cool. Itwas then diluted with ethyl acetate and the mixture washed with waterand the aqueous layer back-extracted with ethyl acetate. The combinedorganic extracts were washed with brine, dried over sodium sulfate,filtered and evaporated. The residue was purified by silica gelchromatography using a gradient of iso-hexanes/ethyl acetate 4:1 to 7:3to afford the title compound (413 mg, 72%) as a beige solid.

To a mixture of 16b (360 mg, 1.73 mmol), potassium vinyltrifluoroborate(279 mg, 2.08 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complexwith dichloromethane (27 mg, 0.033 mmol) in dry n-propanol (27 mL) wasadded triethylamine (175 mg, 241 μL, 1.73 mmol) and the system evacuatedand purged with nitrogen (3×). The reaction mixture was stirred andheated at reflux for 3 h before being allowed to cool. The mixture waspoured into water and extracted with diethyl ether (2×) and the combinedorganic extracts dried over magnesium sulfate, filtered and evaporated.The residue was purified by silica gel chromatography using a gradientof iso-hexanes/ethyl acetate 7:3 to 3:2 to afford the title compound(270 mg, 78%) as a white solid.

To a stirred mixture of 16c (100 mg, 0.500 mmol) and(S)-1-[(S)-2-((S)-2-but-3-enoylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (203 mg, 0.550 mmol) in dichloromethane (10 mL) under nitrogen wasadded N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (134mg, 0.700 mmol) and 4-dimethylaminopyridine (61 mg, 0.500 mmol). Thereaction mixture was stirred for 16 h and then diluted withdichloromethane, washed successively with citric acid solution, waterthen brine, dried over sodium sulfate, filtered and evaporated. Theresidue was purified by silica gel chromatography using a gradient ofiso-hexanes/ethyl acetate 1:1 to 0:1 to afford the title compound (103mg, 38%) as a brown solid.

16d (60 mg, 0.109 mmol) and Hoveyda-Grubbs 2^(nd) generation catalyst(20.5 mg, 0.0328 mmol) was suspended in 1,2-dichloroethane (6 mL) andthe mixture heated in a microwave reactor at 100° C. for 1 h. Theresulting mixture was combined with an identical reaction carried outusing (10 mg, 0.0182) of the(S)-1-[(S)-2-((S)-2-but-3-enoylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (R)-1-(2-vinyl-quinolin-7-yl)-ethyl ester. The volatiles wereevaporated and the residue purified by silica gel chromatography using agradient of ethyl acetate/acetone 1:0 to 4:1 to give a brown gum. Thiswas further purified by preparative thin layer chromatography usingiso-hexanes/ethyl acetate 1:3 to afford the title compound (1.8 mg, 3%)as a white solid. ¹H NMR (300 MHz, CDCl₃) δ0.84-1.02 (m, 6H), 1.26 (apps, 3H), 1.67-1.72 (m, 3H), 1.83-1.96 (m, 1H), 2.00-2.08 (m, 1H),2.63-2.74 (m, 1H), 3.24 (d, J=4.5 Hz, 1H), 3.50 (d, J=7.6 Hz, 1H), 4.21(app t, J=10.3 Hz, 1H), 4.53-4.61 (m, 1H), 5.55 (app t, J=7.3 Hz, 1H),6.04-6.11 (m, 2H), 6.32 (d, J=7.8 Hz, 1H), 6.62 (d, J=16.3 Hz, 1H),6.72-6.80 (m, 1H), 7.29-7.33 (m, 1H), 7.45 (d, J=8.5 Hz, 1H), 7.72 (d,J=8.3 Hz, 1H), 7.97-8.02 (m, 1H). LCMS (m/z) 552.2 [M+H], Tr=1.86 min.

Example 17

Under argon, a solution of diisopropylamine (2.51 g, 24.8 mmol) intetrahydrofuran (150 mL) was cooled in an ice water bath. A solution ofn-butyllithum in hexanes (2.5 M, 9.7 mL, 24 mmol) was added dropwiseover 2 min, and the resulting solution was stirred for 10 additionalmin. The solution was then cooled to −78° C. in a CO₂:acetone bath, andmethyl 2,2-dimethyl-3-oxobutanoate (3.2 g, 22 mmol) was added dropwiseover 30 s. The solution was stirred for an additional 15 min, andN-phenyl-bis(trifluoromethanesulfonimide) (8.4 g, 23.5 mmol) was addedas a solution in tetrahydrofuran (20 mL) via cannula over 5 min, washingwith an additional portion of tetrahydrofuran (10 mL). The resultingsolution was stirred for 10 min and was removed from the cold bath.After stirring an additional 1 h, the reaction mixture was concentratedin vacuo and diluted with diethyl ether (150 mL). The organic phase waswashed with 1 M aqueous sodium hydroxide (1×100 mL, 1×30 mL), dried overanhydrous magnesium sulfate, filtered, and concentrated to afford thetitle compound (6.2 g, 100%) as an amber liquid that was used withoutfurther purification.

A solution of 17a (6.2 g, 22 mmol) in anhydrous pyridine (11 mL, 140mmol) was heated to 70° C. After 18.5 h, the temperature was increasedto 90° C. After stirring for a total of 72 h, the reaction mixture waspartitioned between a stirred mixture of diethyl ether (100 mL) and 3 Maqueous hydrochloric acid (100 mL). The phases were separated, and theorganic layer was washed with saturated aqueous sodium bicarbonate (75mL), dried over magnesium sulfate, filtered, and concentrated to affordthe title compound (2.7 g, 97%) as a slightly brown liquid that was usedwithout further purification.

Under argon, bis(cyclopentadienyl)zirconium chloride hydride (290 mg,1.1 mmol) was cooled in an ice water bath. A solution of 17b (1.4 g,11.1 mmol) and pinacolborane (2.4 mL, 16.5 mmol) in dichloromethane (3mL) was added by cannula, washing with an additional portion ofdichloromethane (2 mL). The resulting mixture was removed from the coldbath and was stirred for 72 h at RT. The reaction was then diluted withethyl acetate (50 mL), quenched with dropwise water (5 mL), and wasfurther diluted with water (50 mL). The organic and aqueous phases wereseparated, and the aqueous phase was extracted with ethyl acetate (30mL). The combined organic phases were dried over anhydrous sodiumsulfate, filtered, and concentrated to afford a crude residue that waspurified by silica gel chromatography (5 to 15% ethyl acetate iniso-hexanes) to afford the title compound (1.6 g, 57%) as a colorlessoil that crystallized on standing at −15° C.

A mixture of 6-bromo-3-chloro-isoquinoline (485 mg, 2.0 mmol),bis(pinacolato)diboron (560 mg, 2.2 mmol), potassium acetate (392 mg,4.0 mmol) and[1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex withdichloromethane (82 mg, 0.1 mmol) in 1,4-dioxane (4 mL) was heated at160° C. in a microwave for 1 hour. The reaction mixture was filteredthrough Celite and the filter pad was washed with ethyl acetate. Thefiltrate was evaporated and the residue was purified by silica gelchromatography using a gradient of dichloromethane todichloromethane/methanol 9:1 to afford the title compound (545 mg, 94%).

A solution of 17d (1.74 g, 6.0 mmol) in tetrahydrofuran (45 mL) wasstirred at 0° C. under nitrogen. Sodium hydroxide solution (2 M, 9 mL,18 mmol) and hydrogen peroxide (30%, 2.5 mL, 24 mmol) were addeddropwise. The reaction mixture was stirred at 0° C. for 30 min. Water(30 mL) was added and the solution was acidified to pH 1 with 2 Mhydrochloric acid. Sodium metabisulfite solution (1 M) was added and themixture was extracted with ethyl acetate. The organic extracts werecombined, washed with water and brine. The organic solution was filteredthrough a hydrophobic frit and the filtrate was evaporated to afford thetitle compound (662 mg, 61%) as an off-white solid.

To a solution of 17e (1.25 g, 7.07 mmol) andtributyl-(1-ethoxy-vinyl)-tin (5.09 g, 4.77 mL, 14.1 mmol) in1,4-dioxane (15 mL) was added bis(triphenylphosphine)palladium(II)dichloride (992 mg, 1.41 mmol) and the reaction mixture was heated at160° C. in a microwave for 30 min. The reaction mixture was filteredthrough Celite and the filter pad was washed with ethyl acetate. Thefiltrate was evaporated and the residue was purified by silica gelchromatography using a gradient of iso-hexanes/ethyl acetate 9:1 to 3:1to afford 3-(1-ethoxy-vinyl)-isoquinolin-6-ol (670 mg) as a gum whichwas used crude in the next step. 3-(1-Ethoxy-vinyl)-isoquinolin-6-ol(670 mg) was suspended in 1,4-dioxane (4 mL) and 4 M hydrogen chloridein 1,4-dioxane (8 mL) was added and the reaction mixture was stirred atRT for 30 min. The solvent was evaporated to afford the title compound(584 mg, 44% over 2 steps) as a white solid.

A solution of 17f (384 mg, 2.05 mmol) and pyridine (0.51 mL, 6.16 mmol)in dichloromethane (30 mL) was stirred in a salt-ice bath for 5 min.Trifluoromethanesulfonic anhydride (0.415 mL, 2.46 mmol) was addeddropwise and the mixture was warmed to RT over 20 min. Additionaltrifluoromethanesulfonic anhydride (0.1 mL, 0.6 mmol) was added and thereaction mixture was stirred at RT for 10 min. Saturated ammoniumchloride solution was added and the mixture was extracted withdichloromethane. The organic extracts were combined, dried over sodiumsulfate, filtered and evaporated. The residue was purified by silica gelchromatography using iso-hexanes/ethyl acetate 19:1 to afford the titlecompound (300 mg, 44%) as a white solid.

To dichloro (p-cymene) ruthenium (II) dimer (2 mg, 0.003 mmol) in water(2 mL) at RT was added (1R,2R)-(−)-N-p-tosyl-1,2-diphenylethylenediamine(3 mg, 0.008 mmol). The system was degassed for 15 min and then heatedto 70° C. for 1.5 h. The reaction was cooled and a solution of 17g (224mg, 0.70 mmol) in degassed anhydrous tetrahydrofuran (1 mL) was addedfollowed by sodium formate (237 mg, 3.50 mmol). The system was degassedfor 2 min and then heated at 40° C. for 1 hour. After cooling to RT,water was added and the water was extracted with dichloromethane (2×).The combined organic layers were dried through a hydrophobic frit andconcentrated in vacuo. The product was purified by silica gelchromatography using iso-hexanes/ethyl acetate 1:1 to afford the titlecompound (220 mg, 97%) as a brown oil.

A round bottom flask was charged with 17h (100 mg, 0.31 mmol),(E)-2,2-dimethyl-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-but-3-enoicacid methyl ester (91 mg, 0.36 mmol),bis[(dicyclohexyl)(4-dimethylaminophenyl)phosphine]palladium(II)chloride (13 mg, 0.02 mmol), potassium phosphate tribasic (198 mg, 0.93mmol) and lithium chloride (40 mg, 0.93 mmol). The system was flushedwith nitrogen and cyclopentyl methyl ether (1 mL) and water (0.5 mL)were added. The reaction was heated for 1 hour at 90° C. and then cooledto RT. Ethyl acetate was added and the solution was washed with water.The organic layer was dried over magnesium sulfate, filtered andconcentrated in vacuo. The residue was purified by silica gelchromatography using iso-hexanes/ethyl acetate 1:2 to give the titlecompound (70 mg, 75%) as a yellow oil.

To 17i (140 mg, 0.47 mmol) in tetrahydrofuran (1 mL), methanol (0.5 mL)and water (0.5 mL) was added lithium hydroxide monohydrate (39 mg, 0.93mmol) at RT. The reaction was stirred for 3 h and quenched by adding 2 Maqueous hydrochloric acid (0.5 mL). The reaction was concentrated invacuo, followed by co-evaporation from methanol and then toluene. Theensuing yellow solid was used crude.

To 1e (250 mg, 0.47 mmol) in anhydrous dichloromethane (5 mL) at 0° C.and under an atmosphere of nitrogen, was added trimethylsilyltrifluoromethanesulfonate (128 μL, 0.70 mmol). The reaction mixture wasstirred at 0° C. for 2 h before adding N,N-diisopropylethylamine (252μL, 1.41 mmol) and then concentrated in vacuo, and co-evaporated withtoluene to afford(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester as a white solid. To 17j (134 mg, 0.47mmol) in anhydrous N,N-dimethylformamide (2 mL) at RT and under anatmosphere of nitrogen was added N,N-diisopropylethylamine (420 μL, 2.35mmol) and 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (250 mg, 0.66 mmol). The solution wasstirred at RT for 3 min before adding a solution of(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester in anhydrous N,N-dimethylformamide (2mL). The reaction was stirred for 16 h. The reaction was quenched withwater and extracted with ethyl acetate (2×). The combined organic layerswere dried over sodium sulfate, filtered and concentrated in vacuo. Theresidue was purified by silica gel chromatography usingiso-hexanes/acetone 1:1 to give the title compound (220 mg, 67% over 3steps) as an off white solid.

To 17k (220 mg, 0.31 mmol) in tetrahydrofuran (3 mL), methanol (1.5 mL)and water (1.5 mL) was added lithium hydroxide monohydrate (53 mg, 1.26mmol) at RT. The reaction was stirred for 1.25 h and quenched by adding2 M aqueous hydrochloric acid (0.64 mL). The reaction was concentratedin vacuo, followed by co-evaporation from methanol (2×) and thenacetonitrile (6×). The resulting residue was dissolved in anhydrousN,N-dimethylformamide (4 mL), and added via syringe pump over 2 h to asuspension of 2-methyl-6-nitrobenzoic anhydride (271 mg, 0.79 mmol),4-dimethylaminopyridine (288 mg, 2.36 mmol) and powdered 4 Å molecularsieves (3 g) in 1,2-dichloroethane (103 mL) at 50° C. Following theaddition the reaction was stirred at 50° C. for 3 h, cooled to RT andfiltered through Celite. The filtrate was concentrated to ⅓ of itsvolume, diluted with dichloromethane and washed with water (2×). Theorganic layer was dried through a hydrophobic frit and concentrated invacuo. The residue was purified by silica gel chromatography usingiso-hexanes/acetone 1:1 to give an off white solid. The solid wastriturated twice with diethyl ether and vacuum dried for 16 h to affordthe title compound (65 mg, 38% over 2 steps) as a white powder. ¹H NMR(300 MHz, CDCl₃) 0.93 (d, J=6.7 Hz, 3H), 0.99 (d, J=6.7 Hz, 3H), 1.41(s, 3H), 1.51 (s, 3H), 1.61 (d, J=7.1 Hz, 3H), 1.76 (d, J=6.7 Hz, 3H),1.76 (m, 1H), 1.78-2.03 (m, 3H), 2.07-2.17 (m, 1H), 2.62-2.76 (m, 1H),3.62-3.79 (m, 2H), 4.21-4.31 (m, 1H), 4.52-4.62 (m, 1H), 5.82-6.01 (m,1H), 6.13 (q, J=6.7 Hz, 1H), 6.27 (d, J=16.1 Hz, 1H), 6.30-6.41 (m, 2H),6.64 (d, J=16.1 Hz, 1H), 7.53 (d, J=8.5 Hz, 1H), 7.71 (s, 1H), 7.82 (s,1H), 7.90 (d, J=8.5 Hz, 1H), 9.16 (s, 1H). LCMS (m/z) 549.9 [M+H],Tr=5.21 min.

Example 18

To Compound 17 (40 mg, 0.073 mmol) in ethyl acetate (10 mL) was added10% palladium on carbon (30 mg) at RT. The system was purged withhydrogen and stirred vigorously for 16 h. The suspension was filteredthrough Celite and the filtrate concentrated in vacuo. The residue waspurified by silica gel chromatography using iso-hexanes/acetone 1:1 toafford Compound 18 (8 mg, 20%) as a white solid. ¹H NMR (300 MHz, CDCl₃)0.92 (d, J=6.5 Hz, 3H), 1.00 (d, J=6.5 Hz, 3H), 1.27 (s, 3H), 1.31 (s,3H), 1.48 (d, J=7.1 Hz, 3H), 1.64-1.79 (m, 1H), 1.75 (d, J=6.7 Hz, 3H),1.80-2.12 (m, 5H), 2.45 (td, J=12.7, 3.6 Hz, 1H), 2.68-2.93 (m, 2H),3.62-3.78 (m, 2H), 3.86 (d, J=12.0 Hz, 1H), 4.37 (app t, J=8.3 Hz, 1H),4.50-4.61 (m, 1H), 5.87-6.00 (m, 1H), 6.18 (q, J=6.7 Hz, 1H), 6.38 (d,J=8.7 Hz, 1H), 6.58 (d, J=8.3 Hz, 1H), 7.36 (d, J=8.5 Hz, 1H), 7.42 (s,1H), 7.64 (s, 1H), 7.83 (d, J=8.5 Hz, 1H), 9.14 (s, 1H). LCMS (m/z)552.2 [M+H], Tr=5.11 min.

Example 19

To a solution of(S)-1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (339 mg, 0.920 mmol) and (R)-1-(3-vinyl-isoquinolin-6-yl)-ethanol(183 mg, 0.920 mmol) in dichloromethane (4.6 mL) were addedN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (211 mg,1.10 mmol) and 4-dimethylaminopyridine (56.2 mg, 0.46 mmol) at 23° C.under an argon atmosphere. After 21 h, the reaction mixture was purifieddirectly by silica gel flash column chromatography to afford the titlecompound (224 mg, 42%) as a light tan solid.

To 19a (800 mg, 1.38 mmol) in anhydrous dichloromethane (12 mL) at 0° C.and under an atmosphere of nitrogen, was added trimethylsilyltrifluoromethanesulfonate (374 μL, 2.07 mmol). The reaction mixture wasstirred at 0° C. for 2 h before adding N,N-diisopropylethylamine (480μL, 2.75 mmol) and then concentrated in vacuo to afford a white solid.To the solid was added a solution of (E)-(R)-2-ethyl-pent-3-enoic acid(188 mg, 1.65 mmol) in anhydrous acetonitrile (12 mL) followed byN,N-diisopropylethylamine (240 μL, 1.38 mmol) and2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uraniumhexafluorophosphate methanaminium (733 mg, 1.93 mmol). The reaction wasstirred at RT for 3 h and concentrated in vacuo. The residue waspurified by silica gel chromatography using iso-hexanes/ethyl acetate1:2 then 1:5 to give the title compound (250 mg, 74% over 2 steps) as aviscous yellow oil.

To 19b (200 mg, 0.34 mmol) in anhydrous toluene (113 mL) at 70° C. andunder an atmosphere of nitrogen, was added Hoveyda-Grubbs 2^(nd)generation catalyst (42 mg, 0.07 mmol). The reaction was heated at 120°C. for 2 h after which an additional amount of the Hoveyda-Grubbs 2^(nd)generation catalyst (30 mg, 0.05 mmol) was added. Following a further 2h at 120° C. the reaction was cooled to RT and potassium isocyanoacetate(83 mg) in methanol (2 mL) was added. The reaction was stirred for 1hour, silica added and then concentrated in vacuo. The residue waspurified by silica gel chromatography using ethyl acetate/acetone 3:1 toafford a brown solid. This was purified further by reverse phasepreparative HPLC to give the title compound (2.2 mg, 1%) as a whitesolid. ¹H NMR (300 MHz, CD₃OD) 0.97 (d, J=6.5 Hz, 3H), 1.02 (d, J=6.5Hz, 3H), 1.11 (t, J=7.4 Hz, 3H), 1.64 (d, J=7.1 Hz, 3H), 1.70 (d, J=6.7Hz, 3H), 1.71-2.06 (m, 7H), 2.66-2.82 (m, 1H), 3.13-3.22 (m, 1H),3.75-3.85 (m, 1H), 4.30-4.48 (m, 2H), 5.51-5.67 (m, 1H), 6.07 (q, J=6.3Hz, 1H), 6.48-6.57 (m, 1H), 6.61 (d, J=16.5 Hz, 1H), 7.47 (s, 1H), 7.58(d, J=8.7 Hz, 1H), 7.89 (s, 1H), 8.07 (d, J=8.7 Hz, 1H), 9.15 (s, 1H).LCMS (m/z) 550.2 [M+H], Tr=1.67 min.

Example 20

To 19a (390 mg, 0.67 mmol) in anhydrous dichloromethane (7 mL) at 0° C.and under an atmosphere of nitrogen, was added trimethylsilyltrifluoromethanesulfonate (182 μL, 1.01 mmol). The reaction mixture wasstirred at 0° C. for 1 hour before quenching with a saturated aqueoussolution of sodium hydrogen carbonate and extracting with ethyl actate(2×). The organic layers were dried over sodium sulfate, filtered andconcentrated in vacuo to afford a white solid. The solid was dissolvedin anhydrous acetonitrile (4 mL) and 2-methyl-but-3-enoic acid (81 mg,0.81 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride(184 mg, 0.94 mmol) and 1-hydroxybenzotriazole monohydrate (103 mg, 0.67mmol) were added. The reaction was stirred at RT for 16 h andconcentrated in vacuo. The residue was purified by silica gelchromatography using iso-hexanes/ethyl acetate 1:1 then 1:3 to give thetitle compound (259 mg, 69%) as a clear viscous oil.

To 20a (250 mg, 0.44 mmol) in degassed anhydrous toluene (148 mL) wasadded 2,6-dichlorobenzoquinone (8 mg, 0.04 mmol). The mixture was heatedto 105° C. and a solution of Hoveyda-Grubbs 2^(nd) generation catalyst(83 mg, 0.13 mmol) in anhydrous toluene (20 mL), was added via syringepump over 2 h. An additional amount of Hoveyda-Grubbs 2^(nd) generationcatalyst (28 mg) was added and the reaction heated to reflux for 1 h.The reaction was cooled to RT, silica gel was added and concentrated invacuo. The residue was purified by silica gel chromatography using ethylacetate/acetone 1:0 then 3:1 to afford an oil. This was triturated withdiethyl ether and a few drops of ethyl acetate to afford a brown solidwhich was further purified by preparative TLC using ethylacetate/acetone 5/1 to give the title compound (9 mg, 1%) as anoff-white solid. ¹H NMR (300 MHz, CDCl₃) 0.97 (d, J=6.7 Hz, 3H), 1.01(d, J=6.7 Hz, 3H), 1.38 (d, J=6.7 Hz, 3H), 1.62 (d, J=7.1 Hz, 3H), 1.70(d, J=6.9 Hz, 3H), 1.72-1.82 (m, 2H), 1.91-2.01 (m, 2H), 2.03-2.12 (m,1H), 2.62-2.74 (m, 1H), 3.35 (app t, J=7.4 Hz, 1H), 3.59 (d, J=12.5 Hz,1H), 3.65-3.77 (m, 1H), 4.26 (app t, J=8.9 Hz, 1H), 4.51-4.60 (m, 1H),5.68-5.79 (m, 1H), 6.06 (q, J=6.7 Hz, 1H), 6.16 (d, J=9.2 Hz, 1H), 6.27(d, J=9.2 Hz, 1H), 6.40 (dd, J=16.0, 7.8 Hz, 1H), 6.71 (d, J=16.0 Hz,1H), 7.39 (dd, J=8.5, 1.3 Hz, 1H), 7.58 (s, 1H), 7.74 (s, 1H), 7.93 (d,J=8.5 Hz, 1H), 9.16 (s, 1H). LCMS (m/z) 536.2 [M+H], Tr=1.63 min.

Example 21

21a was prepared in the same manner as 20a replacing2-methyl-but-3-enoic acid with (R)-2-methyl-pent-4-enoic acid (preparedas described in Synlett 2002, No 12, 2039-2040, 82 mg, 0.72 mmol), toafford the title compound (280 mg, 67%) as a white foam.

To 21a (250 mg, 0.43 mmol) in anhydrous toluene (144 mL) at RT and underan atmosphere of nitrogen, was added Hoveyda-Grubbs 2^(nd) generationcatalyst (54 mg, 0.09 mmol). The reaction was heated at 120° C. for 2 hafter which an additional amount of the Hoveyda-Grubbs 2^(nd) generationcatalyst (25 mg, 0.04 mmol) was added. Following a further 1 h at 120°C. the reaction was cooled to RT and concentrated in vacuo. The residuewas purified by silica gel chromatography using ethyl acetate/acetone1:0 then 3:1 to afford a brown solid. This was triturated with diethylether/ethyl acetate 3:1 to give the title compound (50 mg, 22%) as anoff-white solid. ¹H NMR (300 MHz, CDCl₃) 0.91 (d, J=6.7 Hz, 3H), 0.99(d, J=6.7 Hz, 3H), 1.29 (d, J=7.1 Hz, 3H), 1.59 (d, J=6.7 Hz, 3H), 1.67(d, J=6.5 Hz, 3H), 1.70-1.84 (m, 1H), 1.92-2.15 (m, 4H), 2.33-2.48 (m,1H), 2.63-2.98 (m, 3H), 3.53 (d, J=12.2 Hz, 1H), 3.60-3.72 (m, 1H), 4.42(dd, J=6.7, 2.2 Hz, 1H), 4.52-4.61 (m, 1H), 5.62-5.74 (m, 1H), 6.11 (q,J=6.7 Hz, 1H), 6.16 (q, J=8.9 Hz, 1H), 6.42 (d, J=8.5 Hz, 1H), 6.53-6.66(m, 1H), 6.75 (d, J=16.1 Hz, 1H), 7.37 (dd, J=8.7, 1.0 Hz, 1H),7.80-7.96 (m, 3H), 9.16 (s, 1H). LCMS (m/z) 550.3 [M+H], Tr=1.50 min.

Example 22

To a stirred slurry of 7-bromo-2-chloro-quinoline (8.10 g, 33.4 mmol)and sodium iodide (50.0 g, 334 mmol) in acetonitrile (27 mL) was slowlyadded acetyl chloride (3.56 mL, 50.0 mmol). The flask was stoppered andsealed and heated at 80° C. for 3 h before being allowed to cool. Themixture was treated sequentially with 10% w/w aqueous potassiumcarbonate solution (80 mL), 5% w/w aqueous sodium sulfite solution (80mL) and saturated aqueous sodium thiosulfate solution (80 mL) and themixture extracted with dichloromethane (2×). The combined organicextracts were dried over sodium sulfate, filtered and evaporated to givea crude 7-bromo-2-iodo-quinoline. To the quinoline was addedtributyl(1-ethoxyvinyl)tin (13.6 mL, 40.1 mmol), 1,4-dioxane (67 mL) andbis(triphenylphosphine)palladium(II) dichloride (2.37 g, 3.34 mmol) andthe reaction mixture heated at 100° C. for 5 h before allowing to cool.2 M Aqueous hydrochloric acid (67 mL) was added and the reaction stirredfor 1 h. The mixture was filtered and the solids washed with ethylacetate and the filtrate evaporated to remove organics. The residue wasextracted with ethyl acetate (3×) and the combined organic extracts weredried over sodium sulfate, filtered and evaporated. The product waspurified on silica gel doped with 10% w/w potassium carbonate elutingwith a gradient of 0 to 6% ethyl acetate in iso-hexanes to afford thetitle compound (5.5 g, 66%) as a white solid.

Dichloro (p-cymene) ruthenium(II) dimer (61 mg, 0.100 mmol) and(1R,2R)-(−)-N-p-tosyl-1,2-diphenylethylenediamine (88 mg, 0.012 mmol)was suspended in degassed water (40 mL) and the mixture was degassedwith nitrogen for 5 min. The mixture was stirred at 70° C. undernitrogen for 90 min. The resulting turbid orange mixture was allowed tocool to RT. 22a (5.00 g, 20 mmol) in degassed tetrahydrofuran (40 mL)was added followed by sodium formate (6.8 g, 100 mmol) and the reactionmixture was degassed with nitrogen for 5 min. The reaction mixture wasvigorously stirred at 40° C. for 4 h and allowed to cool. It was thendiluted with ethyl acetate and water and the organic layer wasseparated, washed with water and brine, dried over sodium sulfate,filtered and evaporated. The residue was purified by silica gelchromatography using a gradient of 0% to 30% ethyl acetate iniso-hexanes to afford the title compound (4.96 g, 98%) as an off-whitesolid.

To a solution of 22b (1.00 g, 3.97 mmol) and triethylamine (1.65 mL,11.9 mmol) in anhydrous dichloromethane at 0° C., was added aceticanhydride (0.75 mL, 7.93 mmol) and 4-(dimethylamino)pyridine (24 mg,0.197 mmol). The reaction mixture was stirred and allowed to warm to RT.After 1.5 h water (100 mL) was added and the layers separated. Theaqueous phase was re-extracted with dichloromethane (2×100 mL) and thecombined organics were washed with brine (100 mL), dried over magnesiumsulfate, filtered and concentrated in vacuo to give a crude residue.This was purified by silica gel chromatography using iso-hexanes (66 mL)then iso-hexanes/ethyl acetate 95:5 (300 mL), then iso-hexanes/ethylacetate 9:1 (1066 mL) to yield the title compound (1.16 g, 99%) as acolorless oil.

To a solution of acetic acid 22c (1.16 g, 3.95 mmol) in anhydrousacetonitrile was added palladium(II) acetate (89 mg, 0.395 mmol),2,2-dimethyl-but-3-enoic acid (496 mg, 4.35 mmol), tri(o-tolyl)phosphine(241 mg, 0.790 mmol) and triethylamine (1.09 mL, 7.90 mmol) then themixture was heated in the microwave at 100° C. for 20 min. The reactionmixture was concentrated in vacuo then water (200 mL) was added and theorganics extracted with ethyl acetate (2×50 mL). The combined organicswere dried over sodium sulfate, filtered and concentrated in vacuo togive a crude residue. This was purified by silica gel chromatographyusing iso-hexanes/ethyl acetate 95:5 (300 mL), then iso-hexanes/ethylacetate 9:1 (1066 mL) then iso-hexanes/ethyl acetate 3:1 (1066 mL) toyield the title compound (792 mg, 61%) as a white solid.

A solution of 1e (1.29 g, 2.42 mmol) in dichloromethane (12 mL) wascooled in an ice-water bath under nitrogen. Trimethylsilyltrifluoromethanesulfonate (0.715 mL, 4.84 mmol) was added dropwise, andthe resulting solution was stirred for 1 h. The reaction was quenchedwith N,N-diisopropylethylamine (1.69 mL, 9.68 mmol) and the reactionmixture was concentrated in vacuo to give(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester as a white solid which was used withoutfurther purification. To a solution of 22d (792 mg, 2.42 mmol) inN,N-dimethylformamide (10 mL) at 0° C. under nitrogen was addedN,N-diisopropylethylamine (2.11 mL, 12.1 mmol) and2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (1.01 g, 2.66 mmol). The resultingmixture was stirred at RT for 15 min then re-cooled to 0° C. and(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester generated in the previous step, wasadded as a solution in N,N-dimethylformamide (10.7 mL). The reactionmixture was then allowed to warm to RT with stirring. After 1 h themixture was diluted with ethyl acetate (100 mL) and water (100 mL). Thephases were separated and the aqueous was extracted with ethyl acetate(100 mL). The combined organics were washed with brine (100 mL), driedover magnesium sulfate, filtered and concentrated in vacuo to give acrude residue. This was purified by silica gel chromatography usingiso-hexanes/acetone 95:5 (1000 mL) then iso-hexanes/acetone 9:1 (1 L)then iso-hexanes/acetone 85:15 (1 L) then iso-hexanes/acetone 3:1 (1 L)then iso-hexanes/acetone 7:3 (1 L) to yield the title compound (1.11 g,62%) as a colorless solid.

A solution of 22e (1.11 g, 1.50 mmol) in tetrahydrofuran (14.7 mL) wascooled in an ice-water bath and methanol (7.4 mL), water (7.4 mL), andlithium hydroxide monohydrate (252 mg, 6.0 mmol) were then added. Themixture was stirred for 1 h in the ice-water bath and then quenched withaqueous 1 M hydrochloric acid (6 mL, 6.0 mmol). The resulting solutionwas concentrated in vacuo, and the crude product was concentrated frommethanol (4×250 mL) then acetonitrile (4×250 mL) and toluene (5×250 mL).The solid isolated was then left on the freeze dryer overnight to affordthe title compound (853 mg, quantitative yield) as a white solid.

Under nitrogen, 2-methyl-6-nitrobenzoic anhydride (2.59 g, 7.51 mmol)and 4-(dimethylamino)pyridine (1.38 g, 11.3 mmol) were dissolved in1,2-dichloroethane (500 mL) and the resulting solution was heated to 50°C. The crude seco-acid from the previous step, 22f (853 mg, 1.50 mmol)was dissolved in N,N-dimethylformamide (19 mL) and added to the reactionmixture dropwise via syringe pump over 6 h. After the addition wascomplete, the syringe pump was then rinsed with additionalN,N-dimethylformamide (3 mL) and the reaction mixture was stirred at 50°C. for 40 min. After this time the mixture was concentrated to give aresidue. This was purified by silica gel chromatography usingiso-hexanes (66 mL), then iso-hexanes/acetone 4:1 (726 mL), theniso-hexanes/acetone 7:3 (726 mL), then iso-hexanes/acetone 3:2 (726 mL).This initial column gave impure product (1.10 g) which containedN,N-dimethylformamide. To this was added brine (200 mL) and the organicsextracted with ethyl acetate (2×100 mL). The combined organics weredried over magnesium sulfate, filtered and concentrated in vacuo to giveimpure product (900 mg) which does not contain N,N-dimethylformamide.This residue was purified by a second silica gel chromatography usingiso-hexanes/acetone 95:5 (6 L), then iso-hexanes/acetone 93:7 (1.5 L),then iso-hexanes/acetone 88:12 (3 L), then iso-hexanes/acetone 82:16until all the product eluted from the column. Two batches of the desiredproduct in 87% purity (batch A) and 77% purity (batch B) were isolated.Batch A was triturated twice with 100% diethyl ether to give the titlecompound (241 mg) in approximately 90-95% purity. Purification of BatchB by preparative reverse phase HPLC gave the title compound (181 mg) inapproximately >95% purity. Batch A and B were combined to give the titlecompound (422 mg, 51%) as a white solid. ¹H NMR (500 MHz, CD₃OD) 0.94(app t, J=6.7 Hz, 6H), 1.34 (s, 3H), 1.48 (s, 3H), 1.58 (d, J=7.3 Hz,3H), 1.62-1.75 (m, 5H), 1.82-1.91 (m, 1H), 1.92-2.03 (m, 2H), 2.66-2.78(m, 1H), 3.78 (app d, J=8.5 Hz, 1H), 4.25-4.33 (m, 1H), 4.34-4.43 (m,1H), 5.71 (q, J=7.3 Hz, 1H), 5.89 (q, J=6.7 Hz, 1H), 6.24 (d, J=16.0 Hz,1H), 6.46 (d, J=16.0 Hz, 1H), 7.27 (br d, J=9.8 Hz, 1H), 7.38 (d, J=8.6Hz, 1H), 7.60 (s, 1H), 7.75 (ABq, Δδ_(AB)=0.03, J_(AB)=8.54 Hz, 2H),8.18 (d, J=7.9 Hz, 1H). LCMS (m/z) 550.1 [M+H], Tr=2.24 min.

Example 23

A solution of 6-bromo-3-chloro-isoquinoline (2.0 g, 8.25 mmol) inn-propanol (90 mL) was prepared and potassium vinyltrifluoroborate (1.11g, 8.25 mmol) was added. The solution was purged with nitrogen for 10min before addition of[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complexwith dichloromethane (128 mg, 0.157 mmol) and triethylamine (1.15 mL,8.25 mmol). The reaction mixture was then purged with nitrogen for afurther 3 min before heating to reflux for 1 h. The reaction mixture wasthen allowed to cool to RT and water was added. The organics were thenextracted with diethyl ether (3×150 mL). The combined organics weredried over magnesium sulfate, filtered and concentrated in vacuo to givea crude residue. This was purified by silica gel chromatography, usingiso-hexanes/diethyl ether 9:1 to give the title compound (1.26 g, 80%)as an oil.

A suspension of 23a (1.24 g, 6.54 mmol) in methanol (125 mL) anddichloromethane (125 mL) was cooled to −78° C. The reaction was ozoniseduntil a blue colour persisted (in approximately 15 min), then nitrogenwas bubbled through the reaction mixture for 15 min to purge the ozone.The reaction was then treated with solid sodium bicarbonate (549 mg,6.54 mmol) and dimethyl sulfide (1.31 mL, 1.31 mmol). The mixture wasallowed to warm to RT and after 3 h the reaction mixture wasconcentrated in vacuo. Water (200 mL) was added to the residue and theaqueous layer extracted with dichloromethane (3×200 mL). The combinedorganics were dried over magnesium sulfate, filtered and concentrated invacuo to give the title compound (1.02 g, 81%) as an oil.

To a solution of 23b (1.02 g, 5.32 mmol) in tetrahydrofuran (10 mL) at0° C. was added trimethyl(trifluoromethyl)silane solution (3.19 mL, 2 Min tetrahydrofuran, 6.38 mmol) followed by tetrabutylammonium fluoridesolution (0.053 mL, 1 M in tetrahydrofuran, 0.053 mmol) and theresulting solution was stirred at 0° C. for 1.5 h. The reaction mixturewas then allowed to warm to RT and 2 M hydrochloric acid (10 mL) wasadded and the mixture stirred for 10 min before addition of water (200mL). The aqueous layer was then extracted with ethyl acetate (3×100 mL).The organics were combined, dried over sodium sulfate, filtered andconcentrated to give the title compound (1.30 g, 93%) as a solid.

To a solution of 23c (1.30 g, 4.97 mmol) in dichloromethane (14.7 mL) at0° C. was added Dess-Martin periodinane solution (14.7 mL, 15% indichloromethane, 6.96 mmol) and the reaction mixture was stirred andallowed to warm to RT. 1 M Aqueous sodium metabisulfite (100 mL) wasadded and the reaction mixture was stirred for 15 min. To this mixturewas added saturated aqueous sodium bicarbonate solution (100 mL) and theaqueous layer was extracted with dichloromethane (3×100 mL). Thecombined organics were washed with saturated aqueous sodium bicarbonatesolution (200 mL) followed by brine (100 mL), dried over magnesiumsulfate, filtered and concentrated in vacuo to give the title compound(1.0 g, 78%) as a solid.

A mixture of 23d (686 mg, 2.64 mmol), (R)-2-methyl-2-propanesulfinamide(400 mg, 3.30 mmol) and titanium(IV) isopropoxide (1.95 mL, 6.60 mmol)in tetrahydrofuran (27.4 mL) was heated at reflux for 16 h then allowedto cool to RT. The reaction mixture was concentrated in vacuo andre-dissolved in diethyl ether (27.4 mL) then cooled to −78° C. To thismixture was added L-Selectride® solution (7.93 mL, 1.0 M intetrahydrofuran, 7.93 mmol) and the reaction mixture stirred at −78° C.for 1 h. Brine (30 mL) was then added and the mixture allowed to warm toRT. More brine (200 mL) and ethyl acetate (300 mL) were added and thelayers separated. The aqueous phase was re-extracted withdichloromethane (500 mL). The combined organics were dried over sodiumsulfate, filtered and concentrated in vacuo to give a crude residue.This was purified by silica gel chromatography, using gradient elutionof dichloromethane to dichloromethane/methanol 99:1 to give the titlecompound (553 mg, 57% yield, 94% d.e.).

To a solution of 23e (553 mg, 1.52 mmol) in methanol (10 mL) was added 4M hydrochloric acid solution in 1,4-dioxane (1.52 mL, 6.06 mmol) and thereaction mixture was stirred at RT for 30 min. Additional 4 Mhydrochloric acid solution in 1,4-dioxane (1.52 mL, 6.06 mmol) was addedand the reaction mixture stirred at RT for 30 min then concentrated invacuo to give the title compound (507 mg, quantitative yield).

A mixture of 23f (507 mg, 1.52 mmol) and triethylamine (0.84 mL, 6.08mmol) in dichloromethane (20 mL) was stirred at 0° C. to give asolution. Then a solution of di-tert-butyl dicarbonate (497 mg, 2.28mmol) in dichloromethane (4 mL) was added and the reaction mixture wasstirred and allowed to warm to RT over 24 h. Water (200 mL) was addedand the aqueous layer extracted with dichloromethane (3×100 mL), driedover sodium sulfate, filtered and concentrated in vacuo to give aresidue (816 mg). This residue was dissolved in dichloromethane (20 mL)and cooled to 0° C. then triethylamine (0.84 mL, 6.08 mmol) was addedfollowed by a solution of di-tert-butyl dicarbonate (497 mg, 2.28 mmol)in dichloromethane (4 mL) and the reaction mixture was stirred andallowed to warm to RT over 72 h. Additional di-tert-butyl dicarbonate(162 mg, 0.74 mmol) was added and stirring continued for 30 min thenwater (100 mL) was added and the aqueous layer extracted withdichloromethane (2×50 mL). The combined organics were dried over sodiumsulfate, filtered and concentrated in vacuo to give a crude residue.This was purified by silica gel chromatography, using gradient elutionof iso-hexanes/ethyl acetate 9:1 to iso-hexanes/ethyl acetate 1:1 togive the title compound (140 mg, 25%) as an oil.

To a solution of 23g (135 mg, 0.37 mmol) in n-propanol (6 mL) was addedpotassium vinyltrifluoroborate (55 mg, 0.412 mmol),[1,1′-bis(diphenylphosphino) ferrocene]dichloropalladium(II), complexwith dichloromethane (5.8 mg, 0.007 mmol) and triethylamine (0.052 mL,0.37 mmol). The suspension was evacuated and purged with nitrogen 3times before heating to 100° C. for 2 h. After this time additionalpotassium vinyltrifluoroborate (100 mg, 0.748 mmol) was added andheating continued at 100° C. for 16 h. After this time, additional[1,1′-bis(diphenylphosphino)ferrocene] dichloropalladium(II), complexwith dichloromethane (9 mg, 0.011 mmol) was added and the reactionmixture was heated in a microwave reactor at 150° C. for 90 min. Afterthis time additional[1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex withdichloromethane (9 mg, 0.011 mmol) was added and the reaction mixturewas heated in a microwave reactor at 150° C. for 10 min. Water (100 mL)was added to the reaction mixture and the aqueous layer was thenextracted with diethyl ether (2×100 mL). The combined organics weredried over sodium sulfate, filtered and concentrated in vacuo to givethe title compound (132 mg, quantitative yield).

To a solution of 23h (132 mg, 0.37 mmol) in methanol (2 mL) was added 4M hydrochloric acid in 1,4-dioxane (0.37 mL, 0.75 mmol) and the mixturestirred at RT for 30 min. Additional 4 M hydrochloric acid in1,4-dioxane (3.0 mL, 6.08 mmol) was added and stirring continued for 2 hthen the reaction mixture was concentrated in vacuo to give a residue.The residue was then concentrated from diethyl ether (2 mL), followed byacetonitrile (10 mL) and this was repeated twice to give(S)-2,2,2-trifluoro-1-(3-vinyl-isoquinolin-6-yl)-ethylamine hydrochloricacid salt (108 mg, quantitative yield). To a suspension of(S)-2,2,2-trifluoro-1-(3-vinyl-isoquinolin-6-yl)-ethylamine hydrochloricacid salt (108 mg, 0.374 mmol) in acetonitrile (2 mL) at 0° C. undernitrogen was added(S)-1-[(S)-2-((S)-2-but-3-enoylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (138 mg, 0.37 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (100 mg, 0.52 mmol) and 1-hydroxybenzotriazole hydrate (63mg, 20 wt % in water, 0.37 mmol). The resulting suspension was stirredat 0° C. for 15 min before removing from the ice-water bath and stirringat RT for 16 h. N,N-Diisopropylethylamine (0.5 mL, 2.87 mmol) was addedand the solution was stirred at RT for 2.5 h where additional(S)-1-[(S)-2-((S)-2-but-3-enoylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (69 mg, 0.19 mmol) was added and stirring continued. After 1 h,N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (134 mg,0.70 mmol) was added and the reaction mixture was stirred at RT for 1hour. After this time, the mixture was concentrated in vacuo and theresidue diluted with ethyl acetate (60 mL). Water (50 mL) was added andthe layers separated. The aqueous phase was re-extracted with ethylacetate (2×10 mL) and the combined organics were then washed withammonium chloride (2×50 mL), dried over sodium sulfate, filtered andconcentrated in vacuo to give a crude residue. This was purified bysilica gel chromatography, using gradient elution of iso-hexanes/ethylacetate 9:1 to iso-hexanes/ethyl acetate 4:1 to iso-hexanes/ethylacetate 1:1 and finally iso-hexanes/ethyl acetate 0:1. This gave thetitle compound (124 mg, 55%).

To a solution of 23i (124 mg, 0.21 mmol) in toluene (69 mL) was addedHoveyda-Grubbs 2^(nd) generation catalyst (13 mg, 0.02 mmol) and thereaction mixture heated at 115° C. for 1.5 h. Additional Hoveyda-Grubbs2^(nd) generation catalyst (13 mg, 0.02 mmol) was added and heatingcontinued at 120° C. for 50 min. Additional Hoveyda-Grubbs 2^(nd)generation catalyst (13 mg, 0.02 mmol) was added and heating continuedat 120° C. for 30 min. After this time the mixture was allowed to coolto RT and concentrated in vacuo. The residue was purified by silica gelchromatography using a gradient of ethyl acetate to ethylacetate/acetone 9:1. Impure product (28.3 mg) was collected which wasfurther purified by preparative TLC eluting with ethyl acetate/acetone95:5 to afford the title compound (4 mg, 3%) as a white solid. ¹H NMR(300 MHz, CD₃OD) 0.99 (d, J=6.7 Hz, 3H), 1.01 (d, J=6.7 Hz, 3H),1.29-1.39 (m, 1H), 1.69 (d, J=7.4 Hz, 3H), 1.74-2.01 (m, 5H), 2.73 (td,J=12.9, 2.9 Hz, 1H), 2.99-3.11 (m, 1H), 3.74-3.83 (m, 1H), 4.26 (d,J=10.0 Hz, 1H), 4.41 (br d, J=13.2 Hz, 1H), 5.48 (q, J=7.4 Hz, 1H), 5.84(q, J=8.3 Hz, 1H), 6.58 (d, J=16.3 Hz, 1H), 6.62-6.71 (m, 1H), 7.47 (s,1H), 7.76 (d, J=8.6 Hz, 1H), 7.89 (s, 1H), 8.13 (d, J=8.6 Hz, 1H), 9.22(s, 1H). LCMS (m/z) 575.2 [M+H], Tr=1.53 min.

Example 24

To a solution of (R)-1-(7-bromo-quinolin-2-yl)-ethanol (295 mg, 1.17mmol) in anhydrous acetonitrile (12 mL) were added but-3-enoic acidtert-butyl ester (0.44 mL, 2.75 mmol),bis(tri-tert-butylphosphine)palladium(0) (25 mg, 0.049 mmol) andN,N-dicyclohexylmethylamine (0.39 mL, 1.83 mmol) then the mixture washeated at reflux for 2 h. The reaction mixture was concentrated in vacuoto give a crude residue. This was purified by silica gel chromatographyusing iso-hexanes/ethyl acetate 95:5 to yield the title compound (348mg, 95%) as a white solid.

To 24a (363 mg, 1.16 mmol) and(S)-1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (505 mg, 1.26 mmol) in dichloromethane (20 mL) was addedN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (311 mg,1.62 mmol) and 4-dimethylaminopyridine (142 mg, 1.16 mmol). The reactionmixture was then stirred at RT for 16 h. To the mixture was addedsaturated aqueous ammonium chloride solution (100 mL) and the aqueouslayer extracted with dichloromethane (2×50 mL). The aqueous phase wasfurther washed with dichloromethane (20 mL) and the combined organicswashed with brine (50 mL), dried over magnesium sulfate, filtered andconcentrated in vacuo to give a crude residue. This was purified bysilica gel chromatography using iso-hexanes/ethyl acetate 4:1 (120 mL)then iso-hexanes/ethyl acetate 7:3 (855 mL) then iso-hexanes/ethylacetate 3:2 (540 mL) to yield the title compound (357 mg, 44%) as awhite solid.

To a solution of 24b (342 mg, 0.492 mmol) in dichloromethane (2.5 mL) at0° C. was added trimethylsilyl trifluoromethanesulfonate (0.18 mL, 0.96mmol) and the pale yellow solution stirred at 0° C. for 2.75 h.Additional trimethylsilyl trifluoromethanesulfonate (0.073 mL, 0.45mmol) was added and stirring continued for 1.33 h.N,N-Diisopropylethylamine (0.6 mL, 3.44 mmol) was added and the mixturestirred for 10 min then concentrated in vacuo to give intermediate(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (R)-1-[7-((E)-3-carboxy-propenyl)-quinolin-2-yl]-ethyl ester (266mg, 0.45 mmol). To a solution of(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (R)-1-[7-((E)-3-carboxy-propenyl)-quinolin-2-yl]-ethyl ester (266mg, 0.45 mmol) in acetonitrile (50 mL) at 0° C. under nitrogen was addedN,N-diisopropylethylamine (0.343 mL, 1.971 mmol) and2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (262 mg, 0.70 mmol). The mixture wasstirred at 0° C. and allowed to warm to RT over 16 h. To the reactionmixture was added 2 M aqueous hydrochloric acid solution (20 mL) and themixture concentrated in vacuo. The aqueous layer was extracted withdichloromethane/methanol 9:1 (2×100 mL). The combined organics werewashed with saturated sodium hydrogen carbonate solution (2×200 mL),then brine (1×100 mL), dried over sodium sulfate, filtered andconcentrated in vacuo to give a crude residue. This was purified bysilica gel chromatography using gradient elution of ethylacetate/acetone 97:3 to ethyl acetate/acetone 94:6. Collected 45 mg ofimpure product which was triturated with diethyl ether to give the titlecompound (25.4 mg, 10%) as a pale yellow solid. ¹H NMR (300 MHz, CD₃OD)0.99 (d, J=6.48 Hz, 3H), 1.01 (d, J=6.7 Hz, 3H), 1.64 (d, J=7.1 Hz, 3H),1.66-1.72 (m, 1H), 1.74 (d, J=6.9 Hz, 3H), 1.85-2.08 (m, 4H), 2.75 (dd,J=2.9, 12.9 Hz, 1H), 2.97-3.07 (m, 1H), 3.35-3.40 (m, 1H), 3.79-3.88 (m,1H), 4.23 (d, J=10.5 Hz, 1H), 4.43 (br d, J=11.2 Hz, 1H), 5.72 (q, J=7.1Hz, 1H), 5.94 (q, J=6.9 Hz, 1H), 6.41 (d, J=16.7 Hz, 1H), 6.55 (dt,J=4.7, 16.7 Hz, 1H), 7.40 (d, J=8.5 Hz, 1H), 7.65 (s, 1H), 7.75 (dd,J=1.3, 8.7 Hz, 1H), 7.81 (d, J=8.7 Hz, 1H), 8.21 (d, J=8.5 Hz, 1H). LCMS(m/z) 522.2 [M+H], Tr=1.90 min.

Example 25

A cooled (−20° C.) solution of (S)-6-bromo-indan-1-ol (1.0462 g, 4.910mmol, prepared as described in WO 2009/003719) in anhydroustetrahydrofuran (20 mL) was subsequently treated with triethylamine (2.7mL, 19.639 mmol) and methanesulfonyl chloride (760 μL, 9.820 mmol).After stirring at −30° C. for 2.5 h, a solution of methylamine (2 M intetrahydrofuran, 25 mL, 50 mmol) was added. After stirring at RT for22.5 h the reaction mixture was filtered. The white solid was rinsedwith diethyl ether. The filtrate was evaporated to dryness to afford thetitle compound (1.11 g, quantitative yield) as a white solid.

A solution of 25a (1.11 g, 4.910 mmol) was subsequently treated withdi-tert-butyl dicarbonate (1.714 g, 7.856 mmol) and triethylamine (690μL, 4.91 mmol). After stirring for 25.5 h, the volatiles were removed invacuo and the residue was purified by silica gel chromatography using a50 g Isolute cartridge eluted with a continuous gradient ofiso-hexanes/ethyl acetate 1:0 to 9:1 to afford the title compound(1.4224 g, 89%) as a white solid as a mixture of rotamers.

A solution of 25b (1.4224 g, 4.363 mmol),[1,1-bis(diphenylphosphino)ferrocene] dichloropalladium dichloromethanecomplex (71.3 mg, 0.087 mmol), potassium vinyltrifluoroborate (701.3 mg,5.236 mmol) and triethylamine (610 μL, 4.363 mmol) in anhydrousn-propanol (40 mL) was degassed by bubbling nitrogen through for 30 min.The red suspension was then refluxed for 17.5 h. After cooling to RT,the mixture was quenched with water and the aqueous layer was extractedwith diethyl ether (2×). The organics were combined, dried over sodiumsulfate, filtered and the volatiles were removed in vacuo. The residuewas purified by silica gel chromatography using a 50 g Isolute cartridgeeluted with a continuous gradient of iso-hexanes/diethyl ether 1:0 to4:1 to afford the title compound (977.3 mg, 82%) as a white solid as amixture of rotamers.

A cooled (0° C.) solution of 25c (977.3 mg, 3.575 mmol) indichloromethane (20 mL) was treated with trifluoroacetic acid (5 mL).After stirring for 30 min at RT the volatiles were removed in vacuo andthe residual trifluoroacetic acid was azeotroped off with toluene (3×)to afford the corresponding ammonium salt as a pink solid. A cooled (0°C.) solution of the crude ammonium salt, crude(S)-1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (3.932 mmol) and N,N-diisopropylethylamine (2.5 mL, 14.300 mmol) inacetonitrile (60 mL) was treated with2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (1.903 g, 5.005 mmol). After stirringfor 20 h at RT, the reaction was quenched with hydrochloric acid (1 M,100 mL). The aqueous layer was extracted with ethyl acetate (2×). Theorganics were combined, dried over sodium sulfate, filtered and thevolatiles were removed in vacuo. The residue was purified by silica gelchromatography using a 100 g Isolute cartridge eluted with a continuousgradient of iso-hexanes/ethyl acetate 1:0 to 1:2 to afford the titlecompound in a mixture which was dissolved in ethyl acetate and washedwith aqueous potassium carbonate. The organics were dried over sodiumsulfate, filtered and the volatiles were removed in vacuo to afford thetitle compound (798.9 mg, 36%) as a white solid as a mixture ofrotamers.

Compound 25e was prepared in the same manner as 22e using 25d instead of1e in 27% yield as a complex mixture of rotamers.

A solution of 25e (238.3 mg, 0.390 mmol) in dichloroethane (100 mL) wastreated with Hoveyda-Grubbs 2^(nd) generation catalyst (48.9 mg, 0.078mmol). After stirring at reflux for 1.5 h, the reaction was cooled to RTand the volatiles were removed in vacuo. The residue was purified bysilica gel chromatography using a 10 g Isolute cartridge eluted bygravity with a continuous gradient of ethyl acetate/methanol 1:0 to 95:5followed by preparative TLC eluted with ethyl acetate/methanol 97:3 (2elutions) to provide the final compound (10.2 mg, 5%) as a white solid.¹H NMR (300 MHz, CD₃CN) δ0.85-0.97 (m, 6H), 1.21-1.35 (m, 8H), 1.42-1.62(m, 3H), 1.64-1.77 (m, 2H), 1.80-1.89 (m, 2H), 2.27-2.38 (m, 2H), 2.52(dd, J=7.6, 3.1 Hz, 1H), 2.76 (s, 3H), 2.82-3.06 (m, 2H), 3.27-3.35 (m,1H), 3.47 (s, 3H), 3.80-3.91 (m, 1H), 4.06 (dd, J=9.1, 8.2 Hz, 1H), 4.23(d, J=11.8 Hz, 1H), 4.41 (dd, J=12.9, 3.6 Hz, 1H), 5.44 (app pentet,J=8.0 Hz, 1H), 6.10-6.30 (m, 2H), 6.43 (d, J=16.0 Hz, 1H), 6.88 (d,J=8.0 Hz, 1H), 6.96 (d, J=9.4 Hz, 1H), 7.04 (s, 1H), 7.13-7.28 (m, 2H).LCMS (m/z) 582.3 [M+H], Tr=2.57 min.

Example 26

A solution of(S)-4-benzyl-3-((R)-2-methyl-pent-4-enoyl)-oxazolidin-2-one (1.65 g,6.04 mmol, prepared as in Synlett 2002, 12, 2039-2040) in ethanol/water(22 mL, 10:1) was treated with rhodium(III) chloride hydrate (31.6 mg,0.15 mmol). After stirring at 85° C. for 24 h, the reaction mixture wascooled to RT and the volatiles were removed in vacuo. The residue waspurified by silica gel chromatography using a 50 g Isolute cartridgeeluted with a continuous gradient of iso-hexanes/diethyl ether 1:0 to4:1 to afford the title compound (892.7 mg, 54%) as a colorless oil.

A cooled (0° C.) solution of 26a (893 mg, 3.27 mmol) intetrahydrofuran/water (30 mL, 2:1) was subsequently treated withhydrogen peroxide (1.7 mL, 16.33 mmol, 30% in water) and lithiumhydroxide hydrate (274 mg, 6.53 mmol). After stirring for 1.5 h at 0°C., the reaction was quenched with sodium metabisulfite (6.2 g, 32.66mmol). After stirring at RT for 40 min, the mixture was acidified withhydrochloric acid (2 M) and the aqueous layer was extracted withdichloromethane (2×). The organics were combined, filtered through aphase separator and the volatiles were removed in vacuo. The residue waspurified by silica gel chromatography using a 25 g Isolute cartridgeeluted with a continuous gradient of iso-hexanes/ethyl acetate 1:0 to4:1 to afford the title compound (307.1 mg, 82%) as a colorless oil.

Compound 26c was prepared in the same manner asmethyl-((R)-6-vinyl-indan-1-yl)-carbamic acid tert-butyl ester using(R)-1-(7-bromo-quinolin-2-yl)-ethanol instead of((R)-6-bromo-indan-1-yl)-methyl-carbamic acid tert-butyl ester inquantitative yield.

A solution of(S)-1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (261.2 mg, 0.653 mmol), 26c (108.4 mg, 0.544 mmol) and4-dimethylaminopyridine (79.7 mg, 0.653 mmol) in dichloromethane (10 mL)was treated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (166.9 mg, 0.870 mmol). After stirring at RT for 17 h, thevolatiles were removed in vacuo and the residue was purified by silicagel chromatography using a 50 g Isolute cartridge eluted with acontinuous gradient of iso-hexanes/ethyl acetate 1:0 to 1:4 to 0:1 toafford the title compound (139.6 mg, 44%) as a white solid.

A cooled (0° C.) solution of 26d (139.6 mg, 0.240 mmol) in anhydrousdichloromethane (10 mL) was treated with trimethylsilyl methanesulfonate(90 μL, 0.480 mmol). After stirring for 1.5 h at 0° C., the reaction wasquenched with saturated sodium bicarbonate. The aqueous layer wasextracted with dichloromethane. The organics were combined, filteredthrough a phase separator and the volatiles were removed in vacuo toprovide the intermediate amine. A solution of this amine,(E)-(R)-2-methyl-pent-3-enoic acid (32.9 mg, 0.288 mmol) and1-hydroxybenzotriazole (38.9 mg, 0.288 mmol) in acetonitrile (10 mL) wastreated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (64.4 mg, 0.336 mmol). After stirring at RT for 17.5 h thevolatiles were removed in vacuo and the residue was purified by silicagel chromatography using a 25 g Isolute cartridge eluted with acontinuous gradient of iso-hexanes/ethyl acetate 1:0 to 0:1 to affordthe title compound (69.8 mg, 50%) as a colorless oil.

Compound 26 was prepared in the same manner as compound 25 using 26einstead of(S)-1-{(S)-2-[(S)-2-((2R,3R)-3-methoxy-2-methyl-hept-6-enoylamino)-3-methyl-butyrylamino]-propionyl}-hexahydro-pyridazine-3-carboxylicacid methyl-((R)-6-vinyl-indan-1-yl)-amide in 4% yield. ¹H NMR (300 MHz,CD₃OD) δ0.94-1.00 (m, 6H), 1.47 (d, J=7.6 Hz, 3H), 1.63 (d, J=7.1 Hz,3H), 1.67-1.80 (m, 5H), 1.85-2.07 (m, 3H), 2.76 (td, J=12.1, 3.3 Hz,1H), 3.79-3.86 (m, 1H), 4.28 (d, J=10.5 Hz, 1H), 4.36-4.46 (m, 1H), 5.73(q, J=7.1 Hz, 1H), 5.93 (q, J=6.9 Hz, 1H), 6.32-6.51 (m, 2H), 7.41 (d,J=8.5 Hz, 1H), 7.65 (s, 1H), 7.77-7.85 (m, 2H), 8.21 (d, J=8.5 Hz, 1H).LCMS (m/z) 536.1 [M+H], Tr=1.80 min.

Example 27

A cooled (−78° C.) solution of (of(R)-4-benzyl-3-propionyl-oxazolidin-2-one (3.00 g, 12.9 mmol) inanhydrous tetrahydrofuran (40 mL) was treated with potassiumbis(trimethylsilyl)amide (19.3 mL, 19.3 mmol, 1 M in tetrahydrofuran).After stirring for 0.45 h at −78° C., the mixture was treated with allylbromide (5.6 mL, 64.3 mmol). After stirring for 2 h at −40° C., thereaction was quenched with 2 M hydrochloric acid. The aqueous wasextracted with ethyl acetate (2×50 mL). The organics were combined, andthe volatiles were removed in vacuo. The residue was purified by silicagel chromatography using a 50 g Biotage cartridge eluted with acontinuous gradient of iso-hexanes/ethyl acetate 1:0 to 1:1 to affordthe title compound (2.73 g, 78%) as a colorless oil.

A solution of 27a (2.73 g, 9.97 mmol) in ethanol/water (22 mL, 10:1) wastreated with rhodium(III) chloride hydrate (52 mg, 0.25 mmol). Afterstirring at 85° C. for 3 h, the reaction mixture was cooled to RT andthe volatiles were removed in vacuo. The residue was purified by silicagel chromatography using a 50 g Biotage cartridge eluted with acontinuous gradient of iso-hexanes/ethyl acetate 1:0 to 4:1 to affordthe title compound (1.76 g, 65%) as a colorless oil.

A cooled (0° C.) solution of 27b (1.76 g, 6.44 mmol) intetrahydrofuran/water (60 mL, 2:1) was subsequently treated withhydrogen peroxide (3.3 mL, 32.2 mmol, 30% in water) and lithiumhydroxide hydrate (534 mg, 12.9 mmol). After stirring for 2 h at 0° C.,the reaction was quenched with sodium metabisulfite (12.2 g, 64.4 mmol).After stirring at RT for 1 h, the mixture was acidified with 2 Mhydrochloric acid and the aqueous layer was extracted withdichloromethane (2×100 mL). The organics were combined, filtered and thevolatiles were removed in vacuo. The residue was purified by silica gelchromatography using a 50 g Biotage cartridge eluted with a continuousgradient of iso-hexanes/ethyl acetate 1:0 to 4:1 to afford the titlecompound (463 mg, 63%) as a colorless oil.

A cooled (0° C.) solution of(S)-1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (R)-1-(7-vinyl-quinolin-2-yl)-ethyl ester (380 mg, 0.65 mmol) inanhydrous dichloromethane (10 mL) was treated with trimethylsilylmethanesulfonate (237 μL, 1.31 mmol). After stirring for 1 h at 0° C.,the reaction was quenched with saturated sodium bicarbonate. The aqueouslayer was extracted with dichloromethane. The organics were separatedand combined, volatiles were removed in vacuo to provide theintermediate amine (305 mg). A solution of this amine, 27c (90 mg, 0.76mmol) and 1-hydroxybenzotriazole (103 mg, 0.76 mmol) in acetonitrile (20mL) was treated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (170 mg, 0.89 mmol). After stirring at RT for 16 h thevolatiles were removed in vacuo and the residue was purified by silicagel chromatography using a 25 g Biotage cartridge eluted with acontinuous gradient of iso-hexanes/ethyl acetate 1:0 to 0:1 to affordthe title compound (236 mg, 65%) as a colorless oil.

Compound 27 was prepared in the same manner as compound 25 using 27dinstead of(S)-1-{(S)-2-[(S)-2-((2R,3R)-3-methoxy-2-methyl-hept-6-enoylamino)-3-methyl-butyrylamino]-propionyl}-hexahydro-pyridazine-3-carboxylicacid methyl-((R)-6-vinyl-indan-1-yl)-amide in 5% yield. ¹H NMR (300 MHz,CD₃OD) δ0.85-1.00 (m, 6H), 1.30 (d, J=6.9 Hz, 3H), 1.58 (d, J=7.1 Hz,3H), 1.62-1.80 (m, 6H), 1.83-2.07 (m, 3H), 2.70-2.82 (m, 1H), 3.38-3.48(m, 1H), 3.76-3.84 (m, 1H), 4.20-4.28 (m, 1H), 4.36-4.47 (m, 1H),5.62-5.74 (m, 1H). 5.93 (q, J=6.9 Hz, 1H), 6.30-6.51 (m, 2H), 7.39 (d,J=8.5 Hz, 1H), 7.63 (s, 1H), 7.70-7.80 (m, 2H), 8.13-8.22 (m, 1H). LCMS(m/z) 536.2 [M+H], Tr=2.15 min.

Example 28

Potassium carbonate (647 mg, 4.68 mmol) and di-tert-butyl dicarbonate(716 mg, 3.28 mmol) were added to 2-(3-bromo-phenyl)-piperidine (750 mg,3.12 mmol) in dichloromethane (10 mL). After overnight stirring at RTwater (20 mL) was added to the solution. The resulting biphasic solutionwas separated into organic and aqueous phases. The aqueous phase wasback extracted with dichloromethane (20 mL). The combined organic phaseswere dried over anhydrous sodium sulfate, filtered and concentrated toafford the title compound (1.03 g, 97%) as a pale yellow oil.

To a solution of 28a (1.03 g, 3.03 mmol) and potassiumvinyltrifluoroborate (488 mg, 3.64 mmol) in n-propanol (30 mL), under anatmosphere of nitrogen was added1,1′bis(diphenylphosphino)ferrocenedichloropalladium(II),dichloromethane adduct (49 mg, 0.06 mmol) and triethylamine (306 mg, 422μL, 3.03 mmol). The reaction was heated to reflux and left to stir for 3h before cooling to RT. The reaction mixture poured onto water and theresultant solution was extracted with diethyl ether (3×30 mL). Thecombined organics were dried over anhydrous sodium sulfate, filtered andconcentrated. The resultant residue was purified by silica gelchromatography using a stepwise gradient of iso-hexanes/ethyl acetate1:0 to 4:1 to afford the title compound (652 mg, 75%) as a pale yellowoil.

A solution of(2R,3R)-1-((1R,5S)-10,10-dimethyl-3,3-dioxo-3lambda*6*-thia-4-aza-tricyclo[5.2.1.0*1,5*]dec-4-yl)-3-hydroxy-2-methyl-hept-6-en-1-one(250 mg, 0.703 mmol) in anhydrous dichloromethane (7 mL) was preparedand trimethyloxonium tetrafluoroborate (208 mg, 1.406 mmol) was added.The reaction mixture was stirred at RT for 15 h. The reaction mixturewas treated with methanol (1 mL), then 2 M hydrochloric acid (20 mL) andsaturated brine (20 mL). The mixture was extracted with ethyl acetate(3×15 mL) and the extract was dried over sodium sulfate, filtered andevaporated to give a yellow gum. The gum was purified by silica gelchromatography using iso-hexanes/ethyl acetate 4:1 to give the titlecompound (223 mg, 86%) as a colorless gum.

A solution of 2 M lithium hydroxide in water (5 mL, 10 mmol) was addedto a stirred solution of 28c (223 mg, 0.60 mmol) in tetrahydrofuran (15mL). The stirred mixture was heated to 60° C. for 15 h. The reactionmixture was partially evaporated before adding 2 M hydrochloric acid (20mL). The solution was extracted with ethyl acetate (3×15 mL). Theextract was dried over sodium sulfate, filtered and evaporated to give ayellow gum (209 mg). The gum was purified by silica gel chromatographyusing iso-hexanes/ethyl acetate 3:1 to yield the title compound (68 mg,66%) as a yellow gum.

A solution of(S)-1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methylbutyrylamino)-propionyl]-hexahydropyridazine-3-carboxylicacid 2,2,2-trichloroethyl ester (316 mg, 0.59 mmol) in anhydrousdichloromethane (10 mL) was cooled to 0° C. under a nitrogen atmospherebefore adding trimethylsilyl trifluoromethanesulfonate (160 μL, 0.885mmol). The reaction mixture was stirred at 0° C. for 2 h before addingN,N-diisopropylethylamine (413 μl, 2.36 mmol) to afford(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloroethyl ester. The mixture was evaporated and theresidue dissolved with (2R,3R)-3-methoxy-2-methyl-hept-6-enoic acid (162mg, 0.94 mmol.) in acetonitrile (13 mL) andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (250 mg,1.32 mmol) and 1-hydroxybenzotriazole (220 mg, 1.32 mmol) were added.The reaction was stirred at RT for 15 h then evaporated to give a yellowoil. The oil was purified by silica gel chromatography using ethylacetate to give the title compound (425 mg, 77%) as a white solid.

A solution of 28e (425 mg, 0.725 mmol) in tetrahydrofuran (20 mL) wasprepared and zinc powder (0.48 g, 7.25 mmol) was added followed by anaqueous solution of ammonium acetate (1 M, 5 mL, 5 mmol). The reactionmixture was stirred at RT for 15 h. The reaction was filtered throughhyflo-supercel washing through with ethyl acetate. The mixture wastreated with hydrochloric acid (2 M, 30 mL) and the layers wereseparated. The aqueous layer was extracted with ethyl acetate (25 mL).The organic layers were combined, washed with brine, filtered andevaporated to give a colorless gum (299 mg) which was used directlywithout further purification.

A solution of 2-(3-vinyl-phenyl)-piperidine-1-carboxylic acid tert-butylester (650 mg, 2.26 mmol) in dichloromethane (15 mL) was cooled to 0°C., before adding trimethylsilyl trifluoromethanesulfonate (569 μL, 3.39mmol). The reaction mixture was stirred at 0° C. for 1 h before addingN,N-diisopropylethylamine (1.6 mL, 5.24 mmol) to afford the2-(3-vinyl-phenyl)-piperidine as a yellow solid. The solid wasredissolved, along with 28f (900 mg, 1.98 mmol) in acetonitrile (20 mL).The solution was cooled to 0° C., before adding2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (1.05 g, 2.77 mmol) andN,N-diisopropylethylamine (1.4 mL, 7.92 mmol). The stirred reactionmixture was allowed to slowly warm to RT. After 2 h, the solvent wasevaporated and the remaining residue dissolved in ethyl acetate (30 mL)and washed with water (3×30 mL). The organics were dried over anhydroussodium sulfate, filtered and concentrated. The resultant residue waspurified by silica gel chromatography using a stepwise gradient ofiso-hexanes/ethyl acetate 1:0 to 0:1 to afford the title compound (1.25g, 100%) as a yellow solid.

To a stirred solution of 28g (1.05 mg, 1.68 mmol) in 1,2-dichloroethane(550 mL) was added Hoveyda-Grubbs 2^(nd) generation catalyst (105 mg,0.168 mmol). The solution was heated to 84° C. and was left to stir for2.5 h. The solvent was evaporated and the remaining residue was purifiedby silica gel chromatography using a stepwise gradient ofiso-hexanes/ethyl acetate/acetone 1:0:0 to 0:9:1. This material was thensubjected to a second round of silica gel chromatography using the samegradient to afford a yellow solid. A final round of silica gelchromatography using neat ethyl acetate and eluting purely by gravityafforded the title compound (175 mg, 18%) as a white solid as a 6:4mixture of diastereoisomers. ¹H NMR (300 MHz, CDCl₃) δ0.84 (d, J=6.9 Hz,3H), 0.91-1.01 (m, 6H), 1.24-1.37 (m, 6H), 1.47-2.03 (m, 14H), 2.06 (s,2H), 2.14-2.25 (m, 1H), 2.43-2.77 (m, 4H), 3.48 (s, 1H), 3.56 (s, 2H),3.63-3.86 (m, 1H), 4.00 (app t, J=14.7 Hz, 1H), 4.12-4.17 (m, 1H),4.53-4.65 (m, 1H), 5.45 (q, J=7.2 Hz, 1H), 6.27-6.50 (m, 2H), 6.99-7.10(m, 2H), 7.14-7.22 (m, 1H), 7.32-7.38 (m, 1H). LCMS (m/z) 596.4 [M+H],Tr=2.51 min.

Examples 29 and 30

Compound 28 (155 mg, 0.26 mmol) was dissolved in a 1:1 mixture ofacetonitrile/water to a concentration of 7.8 mg/mL. This solution wasthen eluted through a reverse phase HPLC system fitted with a PhenomenexGemini 10μ 110 A, 250×21.2 mm column using an isocratic 2:3acetonitrile/water flow at 20 mL/min. The mixture was resolved into the2 separate diastereoisomers. On concentration each separatediastereoisomer yielded a white solid. The stereochemistry of eachisomer was not determined. First isomer eluted, Compound 29 (15 mg,10%). ¹H NMR (300 MHz, CDCl₃) δ0.89-1.05 (m, 8H), 1.20-1.44 (m, 10H),1.51-1.79 (m, 3H), 1.83-1.99 (m, 4H), 2.03-2.27 (m, 3H), 2.33-2.67 (m,3H), 2.76 (app t, J=12.3 Hz, 1H), 3.09 (app t, J=12.3 Hz, 1H), 3.22-3.38(m, 1H), 3.47 (s, 3H), 3.54-3.62 (m, 1H), 4.17 (d, J=11.7 Hz, 1H), 4.59(d, J=10.5 Hz, 1H), 5.39-5.53 (m, 1H), 6.01 (s, 1H), 6.09-6.23 (m, 1H),6.30-6.55 (m, 2H), 6.99-7.23 (m, 4H). LCMS (m/z) 596.4 [M+H], Tr=2.53min.

Second isomer eluted, Compound 30 (22 mg, 14%). ¹H NMR (300 MHz, CD₃CN)δ0.98-1.04 (m, 6H), 1.26-1.38 (m, 7H), 1.62-1.80 (m, 5H), 1.83-2.00 (m,4H), 2.03-2.14 (m, 1H), 2.16-2.25 (m, 2H), 2.36-2.51 (m, 2H), 2.54-2.66(m, 1H), 2.68-2.82 (m, 1H), 3.03-3.16 (m, 1H), 3.26-3.34 (m, 1H), 3.48(s, 3H), 3.64-3.73 (m, 1H), 3.76-3.86 (m, 1H), 4.00 (app t, J=9.0 Hz,1H), 4.16 (d, J=12.0 Hz, 1H), 4.55-4.66 (m, 1H), 5.41-5.53 (m, 1H),5.97-6.06 (m, 1H), 6.10-6.22 (m, 1H), 6.35 (s, 1H), 6.38-6.49 (m, 1H),7.00-7.11 (m, 2H), 7.14-7.23 (m, 3H). LCMS (m/z) 596.3 [M+H], Tr=2.49min.

Example 31

A cooled (0° C.) solution of(R)-4-benzyl-3-[(R)-2-((S)-1-hydroxy-ethyl)-but-3-enoyl]-oxazolidin-2-one(233.6 mg, 0.807 mmol, prepared as described in Org. Lett. 2007, 9,1635-1638) and imidazole (241.7 mg, 3.551 mmol) in N,N-dimethylformamide(2 mL) was treated with tert-butyldimethylsilyl chloride (158.2 mg,1.049 mmol). After stirring for 24 h at RT, the reaction was quenchedwith saturated ammonium chloride. The aqueous layer was extracted withdiethyl ether (2×20 mL). The organics were combined, dried over sodiumsulfate, filtered and the volatiles were removed in vacuo. The residuewas purified by silica gel chromatography using a 25 g Isolute cartridgeeluted with a continuous gradient of iso-hexanes/ethyl acetate 1:0 to4:1 to afford the title compound (294.6 mg, 90%) as a colorless oil.

A solution of 31a (294.6 mg, 0.730 mmol),(R)-1-(7-bromo-quinolin-2-yl)-ethanol (184.0 mg, 0.730 mmol),palladium(II)acetate (32.8 mg, 0.146 mmol), tri-(o-toluyl)phosphine(44.4 mg, 0.146 mmol) in anhydrous 1,4-dioxane (10 mL) was treated withN,N-dicylohexylmethylamine (250 μL, 1.168 mmol). After stirring at 100°C. for 5 h, the reaction was cooled to RT, diluted with dichloromethaneand saturated sodium bicarbonate. The aqueous layer was extracted withdichloromethane. The organics were combined, filtered through a phaseseparator and the volatiles were removed in vacuo. The residue waspurified by silica gel chromatography using a 25 g Isolute cartridgeeluted with a continuous gradient of iso-hexanes/ethyl acetate 1:0 to7:3 to afford the title compound (230.8 mg, 55%) as a colorless oil.

A cooled (0° C.) solution of 31b (230.8 mg, 0.401 mmol) intetrahydrofuran/water (15 mL, 2:1) was subsequently treated withhydrogen peroxide (30% aqueous, 210 μL, 2.005 mmol) and lithiumhydroxide hydrate (33.7 mg, 0.803 mmol). After stirring for 2 h at 0°C., the reaction was quenched with sodium metabisulfite (765 mg, 4.1mmol). After stirring for 3.5 h at RT the volatiles were removed invacuo. The mixture was then diluted with water and the pH was adjustedwith potassium carbonate. The aqueous layer was washed withdichloromethane (2×20 mL) and acidified with 2 M hydrochloric acid (pH˜1) then extracted with dichloromethane (3×30 mL). All the organics werecombined, dried over sodium sulfate, filtered and the volatiles wereremoved in vacuo. The residue was purified by silica gel chromatographyusing a 25 g Isolute cartridge eluted with a continuous gradient ofiso-hexanes/ethyl acetate 1:0 to 0:1 to afford the title compound (69.8mg, 42%) as a white solid.

Compound 31d was prepared in the same manner as 22e using 31c instead of(E)-4-[2-((R)-1-acetoxy-ethyl)-quinolin-7-yl]-2,2-dimethyl-but-3-enoicacid in 46% yield.

Compound 31 was prepared in the same manner as Compound 22 using 31dinstead of(S)-1-[(S)-2-((S)-2-{(E)-4-[2-((R)-1-hydroxy-ethyl)-quinolin-7-yl]-2,2-dimethyl-but-3-enoylamino}-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester in 10% yield. ¹H NMR (300 MHz, CDCl₃)δ1.00 (d, J=6.7 Hz, 3H), 1.01 (d, J=6.7 Hz, 3H), 1.22-1.44 (m, 4H), 1.64(d, J=7.1 Hz, 3H), 1.68-1.77 (m, 4H), 1.89-2.08 (m, 3H), 2.71-2.84 (m,1H), 3.78-3.86 (m, 1H), 4.11 (dd, J=8.2, 6.2 Hz, 1H), 4.17-4.25 (m, 2H),4.38-4.47 (m, 1H), 5.68 (q, J=7.1 Hz, 1H), 5.94 (q, J=6.9 Hz, 1H), 6.47(d, J=16.5 Hz, 1H), 6.69 (dd, J=16.5, 5.3 Hz, 1H), 7.40 (d, J=8.5 Hz,1H), 7.68 (s, 1H), 7.76 (dd, J=8.7, 1.3 Hz, 1H), 7.81 (d, J=8.7 Hz, 1H),8.21 (d, J=8.5 Hz, 1H). LCMS (m/z) 566.1 [M+H], Tr=1.65 min.

Example 32

To a cooled (0° C.) solution of ethyl propiolate (2 mL, 19.735 mmol) indichloromethane (50 mL) was added dropwise morpholine (1.7 mL, 19.435mmol). After stirring at RT for 1.5 h, the volatiles were removed invacuo and the residue was purified by silica gel chromatography using a50 g Isolute cartridge eluted with dichloromethane/methanol 20:1 toafford the title compound (3.5034 g, 97%) as a colorless oil.

A cooled (0° C.) solution of 4-bromoaniline (2.7 g, 15.7 mmol) in water(30 mL) was subsequently treated with concentrated hydrochloric acid(3.5 mL) and sodium nitrite (1.3 g, 18.840 mmol). After 20 min at 0° C.,concentrated hydrochloric acid (5.3 mL) and sodium tetrafluoroborate(6.9 g, 62.847 mmol) were added. After 40 min at 0° C., the intermediatediazonium was filtered, washed with water, methanol and diethyl ether(2.1021 g) and was used without further purification. A solution of thediazonium (7.762 mmol) in acetonitrile (50 mL) was treated with 32a(1.6614 g, 8.970 mmol). After 1 h at RT, silica gel was added. Afterstirring at RT for 16 h, the volatiles were removed in vacuo and theresidue was purified by silica gel chromatography using a 100 g Isolutecartridge eluted with a continuous gradient of iso-hexanes/ethyl acetate1:0 to 9:1 to afford the title compound (1.9811 g, 85%) as a highlycoloured solid and as a mixture of tautomers.

A solution of 32b (1.9811 g, 6.623 mmol) in concentrated sulphuric acid(25 mL) was heated at 100° C. for 3 h. After cooling to 0° C., themixture was diluted with water (150 mL) and a brown solid was filteredoff. The filtrate was extracted with diethyl ether (2×50 mL),dichloromethane (2×50 mL) and ethyl acetate (50 mL). The organics werecombined, dried over sodium sulfate, filtered and the volatiles wereremoved in vacuo to provide the title compound (1.5094 g, 90%) as anorange solid that turned dark upon standing.

A cooled (0° C.) solution of 32c (1.5094 g, 5.964 mmol),N,O-dimethylhydroxylamine hydrochloride (755.9 mg, 7.753 mmol) andN,N-diisopropylethylamine (4.2 mL, 23.856 mmol) in acetonitrile (50 mL)was treated with 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methanaminium (3.175 g, 8.350 mmol). Afterstirring for 6 h at RT, the mixture was cooled to 0° C. and quenchedwith 1 M hydrochloric acid (60 mL). The aqueous layer was extracted withethyl acetate (3×50 mL). The organics were combined, dried over sodiumsulfate, filtered and the volatiles were removed in vacuo. The residuewas purified by silica gel chromatography using a 100 g Isolutecartridge eluted with a continuous gradient of iso-hexanes/ethyl acetate1:0 to 1:1 to afford the title compound (1.4229 g, 81%) as a brightyellow solid.

A cooled (−78° C.) solution of 32d (1.4229 g, 4.805 mmol) intetrahydrofuran (50 mL) was treated with methylmagnesium chloride (3.2mL, 9.610 mmol, 3 M in diethyl ether). After 1 h at −78° C. and 3 h at0° C., the reaction was quenched with saturated ammonium chloride (30mL). The aqueous layer was extracted with ethyl acetate (3×50 mL). Theorganics were combined, dried over sodium sulfate, filtered and thevolatiles were removed in vacuo. The residue was purified by silica gelchromatography using a 100 g Isolute cartridge eluted with a continuousgradient of iso-hexanes/ethyl acetate 1:0 to 4:1 to afford the titlecompound (751.5 mg, 62%) as a yellow solid.

A solution of 32e (751.5 mg, 2.993 mmol) in tetrahydrofuran (30 mL) wastreated with (S)-(−)-2-methyl-CBS-oxazaborolidine (3.6 mL, 3.592 mmol, 1M in toluene). After 10 min at RT the mixture was cooled to −60° C. andtreated with borane-tetrahydrofuran complex (6 mL, 5.986 mmol, 1 M intetrahydrofuran). After 1.5 h at −55° C. to −30° C., the reaction wasquenched with methanol (20 mL). After stirring at RT for 16 h, thevolatiles were removed in vacuo. The residue was purified by silica gelchromatography using a 50 g Isolute cartridge eluted with a continuousgradient of iso-hexanes/ethyl acetate 1:0 to 3:2 to afford the titlecompound (345.0 mg, 45%) as a yellow solid.

A solution of 32f (345.0 mg, 1.363 mmol), palladium II acetate (61.2 mg,0.273 mmol), tri-(o-toluyl)phosphine (83.1 mg, 0.273 mmol), 3-butenoicacid tert-butyl ester (560 μL, 3.407 mmol) andN,N-dicyclohexylmethylamine (470 μL, 2.181 mmol) in anhydrous1,4-dioxane (20 mL) was heated at 100° C. for 1.7 h. After cooling to RTthe volatiles were removed in vacuo. The residue was purified by silicagel chromatography using a 50 g Isolute cartridge eluted with acontinuous gradient of iso-hexanes/ethyl acetate 1:0 to 2:3 to affordthe title compound (244.1 mg, 57%) as a yellow solid.

A cooled (0° C.) solution of 1e (422.0 mg, 0.793 mmol) intetrahydrofuran/water (25 mL, 4:1) was treated with lithium hydroxidehydrate (67.0 mg, 1.587 mmol). After stirring at 0° C. for 1.5 h, thereaction was quenched with 1 M hydrochloric acid (20 mL). The aqueouslayer was extracted with ethyl acetate (2×30 mL). The organics werecombined, dried over sodium sulfate, filtered and the volatiles wereremoved in vacuo then residual trichloroethanol was azeotroped off withtoluene (3×) to give the intermediate acid as a white solid which wasthen combined with 32g (244.1 mg, 0.793 mmol), 4-dimethylaminopyridine(97.0 mg, 0.793 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (243.3 mg, 1.269 mmol) and dichloromethane (20 mL). Afterstirring at RT for 16 h, the reaction was quenched with dilutehydrochloric acid. The aqueous layer was extracted with dichloromethane(30 mL). The organics were combined and filtered through a phaseseparator and the volatiles were removed in vacuo. The residue waspurified by silica gel chromatography using a 50 g Isolute cartridgeeluted with a continuous gradient of iso-hexane/sethyl acetate 1:0 to1:2 to afford the title compound (164.4 mg, 30% over 2 steps) as ayellow glass.

A cooled (0° C.) solution of 32h (164.4 mg, 0.236 mmol) in anhydrousdichloromethane (20 mL) was treated with trimethylsilyl methanesulfonate(170 μL, 0.944 mmol). After stirring for 1 h at 0° C., the reaction wasquenched with N,N-diisopropylethylamine (330 μL, 1.888 mmol) and thevolatiles were removed in vacuo. A cooled (0° C.) solution of the crudeamino acid in acetonitrile (80 mL) was subsequently treated withN,N-diisopropylethylamine (330 μL, 1.888 mmol) and2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (125.6 mg, 0.330 mmol). After stirringat RT for 2.5 h, the volatiles were removed in vacuo. The residue waspurified by silica gel chromatography using a 50 g Isolute cartridgeeluted with a continuous gradient of iso-hexanes/ethyl acetate 1:0 to0:1 to afford the final compound (170.7 mg) as a mixture. Reverse phasepreparative HPLC which was eluted with a gradient of water/acetonitrile95:5 to 0:100 provided the title compound (30.6 mg, 25%) as a whitesolid. ¹H NMR (300 MHz, CD₃OD) δ1.00 (d, J=6.7 Hz, 3H), 1.03 (d, J=6.7Hz, 3H), 1.61 (d, J=7.3 Hz, 3H), 1.66-2.07 (m, 8H), 2.68-2.81 (m, 1H),2.97-3.07 (m, 1H), 3.35-3.47 (m, 1H), 3.76-3.89 (m, 1H), 4.28 (d, J=9.4Hz, 1H), 4.37-4.47 (m, 1H), 4.53-4.67 (m, 1H), 5.52 (q, J=7.1 Hz, 1H),6.36-6.51 (m, 2H), 6.72 (d, J=16.0 Hz, 1H), 7.61 (s, 1H), 7.97 (dd,J=8.9, 1.6 Hz, 1H), 8.06 (s, 1H), 8.38 (d, J=8.9 Hz, 1H). LCMS (m/z)523.1 [M+H], Tr=1.65 min.

Examples 33 and 34

A cooled (0° C.) solution of(S)-tetrahydro-pyridazine-1,2,3-tricarboxylic acid 1,2-di-tert-butylester 3-(2,2,2-trichloro-ethyl) ester (5.5297 g, 11.975 mmol) intetrahydrofuran/methanol (50 mL, 1:1) was treated withtetrabutylammonium fluoride (23.9 mL, 23.950 mmol, 1 M intetrahydrofuran). After stirring at RT for 24 h, the volatiles wereremoved in vacuo. The residue was dissolved in diethyl ether andsaturated sodium bicarbonate. The aqueous layer was extracted withdiethyl ether (50 mL). The organics were combined, dried over sodiumsulfate, filtered and the volatiles were removed in vacuo. The residuewas purified by silica gel chromatography using a 100 g Isolutecartridge eluted with a continuous gradient of iso-hexanes/ethyl acetate1:0 to 9:1 where the mixed fractions were further purified by silica gelchromatography using a 50 g Isolute cartridge eluted with a continuousgradient of iso-hexanes/ethyl acetate 1:0 to 4:1 to afford the titlecompound (3.9765 g, 96%) as a colorless oil and as a mixture ofrotamers.

A cooled (−78° C.) solution of 33a (1.4112 g, 4.097 mmol) in anhydroustetrahydrofuran (10 mL) was treated with lithiumbis(trimethylsilyl)amide (6.2 mL, 6.146 mmol, 1 M in tetrahydrofuran).After stirring for 1.25 h at −78° C., the mixture was treated withiodomethane (640 μL, 10.242 mmol). After stirring for 1 h at −78° C., 1h at 0° C. and 1 h at RT, the reaction was quenched with pH 7 buffer at0° C. The aqueous was extracted with dichloromethane (2×50 mL). Theorganics were combined, dried over sodium sulfate, filtered and thevolatiles were removed in vacuo. The residue was purified by silica gelchromatography using a 50 g Isolute cartridge eluted with a continuousgradient of iso-hexanes/ethyl acetate 1:0 to 4:1 to afford the titlecompound (760.7 mq, 52%) as a colorless oil and as a mixture ofrotamers.

A cooled (0° C.) solution of(S)-2-tert-butoxycarbonylamino-3-methyl-butyric acid (3.020 g, 13.908mmol), (S)-2-amino-propionic acid methyl ester hydrochloride (1.9413 g,13.908 mmol) and N,N-diisopropylethylamine (17.1 mL, 55.632 mmol) inacetonitrile (50 mL) was treated with2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (7.403 g, 19.471 mmol). After stirringat RT for 6 h, the reaction was quenched with hydrochloric acid (1 M,100 mL). The aqueous layer was extracted with ethyl acetate (2×100 mL).The organics were combined, dried over sodium sulfate, filtered and thevolatiles were removed in vacuo. The residue was purified by silica gelchromatography using a 100 g Isolute cartridge eluted with a continuousgradient of iso-hexanes/ethyl acetate 1:0 to 3:2 to afford the titlecompound (4.0996 g, 97%) as a white solid.

A cooled (0° C.) solution of 33c (317.0 mg, 1.048 mmol) intetrahydrofuran/water (12 mL, 5:1) was treated with lithium hydroxidehydrate (88.0 mg, 2.096 mmol). After stirring at 0° C. for 2.5 h, thereaction was quenched with hydrochloric acid (1 M, 30 mL). The aqueouslayer was extracted with dichloromethane (2×30 mL). The organics werecombined, filtered through a phase separator and the volatiles wereremoved in vacuo to provide crude acid as a white solid.

A cooled (0° C.) solution of 33b (376.0 mg, 1.049 mmol) in anhydrousdichloromethane (15 mL) was treated with trifluoroacetic acid (5 mL).After stirring at 0° C. for 30 min and RT for 2 h, trifluoroacetic acid(4 mL) was added. After 1 h, the volatiles were removed in vacuo and theresidual trifluoroacetic acid azeotroped off with toluene (3×) toprovide the bis-trifluoroacetic acid ammonium salt as an off-whitesolid. A cooled (0° C.) solution of the bis-trifluoroacetic acidammonium salt and crude(S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-propionicacid as prepared in the previous step in anhydrous dichloromethane (15mL) was subsequently treated with N-methylmorpholine (580 μL, 5.240mmol) and O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (596.2 mg, 1.572 mmol). After stirring for 19 h atRT, the reaction was quenched with hydrochloric acid (1 M, 30 mL). Theaqueous was extracted with dichloromethane (2×30 mL). The organics werecombined, washed with saturated aqueous sodium bicarbonate and filteredthrough a phase separator. The volatiles were removed in vacuo and theresidue was purified by silica gel chromatography using a 50 g Isolutecartridge eluted with a continuous gradient of iso-hexanes/ethyl acetate1:0 to 1:2 to afford the title compound (373.1 mg, 83% over 2 steps) asa white foam and as a mixture of diastereoisomers.

A cooled (0° C.) solution of 33e (373.1 mg, 0.871 mmol) in anhydrousdichloromethane (10 mL) was treated with trimethylsilyl methanesulfonate(310 μL, 1.741 mmol). After stirring for 1 h at 0° C., the reaction wasquenched with N,N-diisopropylethylamine (610 μL, 3.484 mmol) and thevolatiles were removed in vacuo. A cooled (0° C.) solution of the crudeamine and (E)-(2R,3R)-3-methoxy-2-methyl-hex-4-enoic acid (137.8 mg,0.871 mmol) in anhydrous acetonitrile (15 mL) was subsequently treatedwith N,N-diisopropylethylamine (610 μL, 3.484 mmol) and2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (463.7 mg, 1.219 mmol). After stirringat RT for 18 h, the reaction was quenched with hydrochloric acid (1 M,30 mL). The aqueous layer was extracted with ethyl acetate (2×30 mL).The organics were combined, washed with saturated sodium bicarbonate (20mL), dried over sodium sulfate, filtered and the volatiles were removedin vacuo. The residue was purified by silica gel chromatography using a25 g Isolute cartridge eluted with a continuous gradient ofiso-hexanes/ethyl acetate 1:0 to 1:2 to 1:4 to afford the title compound(332.2 mg, 81%) as a colorless solid and as a mixture ofdiastereoisomers.

Compound 33g was prepared in the same manner as(S)-1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (R)-1-[6-((E)-3-tert-butoxycarbonyl-propenyl)-cinnolin-3-yl]-ethylester using 33f and (R)-1-(3-vinyl-isoquinolin-6-yl)-ethanol instead of1e and (E)-4-[3-((R)-1-hydroxy-ethyl)-cinnolin-6-yl]-but-3-enoic acidtert-butyl ester in 30% yield over 2 steps.

A solution of 33g (136.2 mg, 0.214 mmol) in toluene (70 mL) was degassedby bubbling N₂ gas through for 20 min. Hoveyda-Grubbs 2^(nd) generationcatalyst (27 mg, 0.043 mmol) was added and the mixture was refluxed for2.5 h. More Hoveyda-Grubbs 2^(nd) generation catalyst (20 mg) was addedand after stirring at reflux for 1 h, the mixture was cooled to RT, thevolatiles were removed in vacuo and the residue was purified by silicagel chromatography using a 25 g Isolute cartridge eluted with acontinuous gradient of ethyl acetate/acetone 1:0 to 10:1 to afford bothdiastereoisomers in separate mixtures. Both mixtures were purified bysilica gel chromatography using a 10 g Isolute cartridge eluted bygravity with a continuous gradient of ethyl acetate/acetone 1:0 to 10:1to provide the more polar diastereoisomer (19.7 mg, 15%) as a whitesolid. The less polar diastereomer was further purified by preparativeTLC eluted with ethyl acetate (4 elutions) and subsequent preparativeTLC eluted with iso-hexanes/acetone 7:3 (2 elutions) to afford the titlecompound (7.6 mg, 6%) as a white solid. Relative stereochemistry was notassigned.

More polar diastereoisomer Compound 33: ¹H NMR (300 MHz, CDCl₃)δ0.83-1.07 (m, 7H), 1.41 (d, J=7.1 Hz, 3H), 1.47 (d, J=6.9 Hz, 3H), 1.50(s, 3H), 1.70 (d, J=6.7 Hz, 3H), 1.75-1.90 (m, 2H), 1.96-2.15 (m, 3H),2.64 (dd, J=7.6, 3.1 Hz, 1H), 2.70-2.84 (m, 1H), 3.42 (s, 3H), 3.90 (dd,J=8.7, 3.1 Hz, 1H), 4.00 (app t, J=8.0 Hz, 1H), 4.10 (s, 1H), 4.58-4.69(m, 1H), 6.00 (dd, J=9.4, 7.1 Hz, 1H), 6.22 (q, J=6.2 Hz, 1H), 6.51 (d,J=9.8 Hz, 1H), 6.93 (s, 1H), 7.13-7.23 (m, 1H), 7.34-7.43 (m, 1H),7.90-7.97 (m, 2H), 8.47 (s, 1H). LCMS (m/z) 594.3 [M+H], Tr=1.87 min.

Less polar diastereoisomer Compound 34: ¹H NMR (300 MHz, CD₃CN)δ−0.51-−0.39 (m, 5H), −0.30 (d, J=6.9 Hz, 3H), 0.06 (d, J=7.1 Hz, 3H),0.15 (d, J=6.9 Hz, 3H), 0.19 (s, 3H), 0.25-0.34 (m, 4H), 0.38-0.45 (m,1H), 0.81-1.02 (m, 2H), 1.14-1.42 (m, 2H), 2.69 (dd, J=8.7, 2.9 Hz, 1H),3.00-3.10 (m, 2H), 3.45 (s, 3H), 4.32 (app pentet, J=7.1 Hz, 1H), 4.76(q, J=6.7 Hz, 1H), 5.53 (d, J=16.3 Hz, 1H), 5.85 (dd, J=16.1, 8.9 Hz,1H), 6.20 (dd, J=8.5, 1.6 Hz, 1H), 6.41 (d, J=8.9 Hz, 1H), 6.65-6.75 (m,2H), 6.91 (s, 1H), 7.11 (d, J=7.3 Hz, 1H), 7.79 (s, 1H). LCMS (m/z)594.3 [M+H], Tr=1.83 min.

Example 35

A cooled (0° C.) solution of 2-fluoro-5-hydroxy-benzoic acid (1.0051 g,6.438 mmol), N,O-dimethylhydroxylamine hydrochloride (1.2560 g, 12.856mmol) and triethylamine (3.6 mL, 25.752 mmol) in dichloromethane (35 mL)was treated with O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (3.663 g, 9.658 mmol). After stirring at RT for 20h, the reaction was quenched at 0° C. with hydrochloric acid (2 M, 30mL). The emulsion was filtered on Celite then the aqueous layer wasextracted with dichloromethane (50 mL). The organics were combined,filtered through a phase separator and the volatiles were removed invacuo. The residue was purified by silica gel chromatography using a 50g Isolute cartridge eluted with a continuous gradient of toluene/diethylether 1:0 to 1:2 to afford the title compound (260.5 mg, 20%) as acolorless oil.

A solution of 35a (260.5 mg, 1.308 mmol) in acetone (20 mL) wassubsequently treated with potassium carbonate (903.8 mg, 6.539 mmol) andallylbromide (340 μL, 3.924 mmol). After stirring at RT for 24 h, thereaction was quenched with water (20 mL). The aqueous layer wasextracted with dichloromethane (2×30 mL). The organics were combined,filtered through a phase separator and the volatiles were removed invacuo. The residue was purified by silica gel chromatography using a 25g Isolute cartridge eluted with a continuous gradient ofiso-hexanes/ethyl acetate 1:0 to 3:2 to afford the title compound (289.7mg, 92%) as a colorless oil.

A cooled (−78° C.) solution of 35b (289.7 mg, 1.210 mmol) intetrahydrofuran (10 mL) was treated with methylmagnesium chloride (810μL, 2.422 mmol, 3 M in diethyl ether). After 1.25 h at −78° C., 2 h at0° C. and 16 h at RT, methylmagnesium chloride (810 μL, 2.422 mmol, 3 Min diethyl ether) was added. After 2.5 h at −78° C. and 1.2 h at RT, thereaction was quenched with silica gel and the volatiles were removed invacuo. The residue was purified by silica gel chromatography using a 25g Isolute cartridge eluted with a continuous gradient ofiso-hexanes/ethyl acetate 1:0 to 9:1 to afford the title compound (196.7mg, 84%) as a colorless oil.

A solution of 35c (196.7 mg, 1.013 mmol) in tetrahydrofuran (15 mL) wastreated with (S)-(−)-2-methyl-CBS-oxazaborolidine (1.2 mL, 1.215 mmol, 1M in toluene). After 10 min at RT the mixture was cooled to −50° C. andtreated with borane tetrahydrofuran complex (2.1 mL, 2.026 mmol, 1 M intetrahydrofuran). After 1.5 h at −50° C. to −40° C., the reaction wasquenched with methanol (6 mL). After stirring at RT for 22 h, thevolatiles were removed in vacuo. The residue was purified by silica gelchromatography using a 25 g Isolute cartridge eluted with a continuousgradient of iso-hexanes/ethyl acetate 1:0 to 4:1 to afford the titlecompound (179.4 mg, 90%) as a white solid.

A cooled (0° C.) solution of 1e (1.9973 g, 3.755 mmol) in anhydrousdichloromethane (40 mL) was treated with trimethylsilyltrifluoromethanesulfonate (1.4 mL, 7.510 mmol). After 30 min at 0° C.,the reaction mixture was treated with N, N-diisopropylethylamine (2.6mL, 15.020 mmol) and the volatiles were removed in vacuo to afford thecorresponding amine. A cooled (0° C.) solution of this amine,(E)-(2R,3R)-2-methylhex-6-enoic acid (428.6 mg, 3.755 mmol, prepared asdescribed in Synlett 2002, 12, pp 2039-2040) and N,N-diisopropylethylamine (2.6 mL, 15.020 mmol) in acetonitrile (50 mL)was treated with 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methanaminium (1.999 g, 5.257 mmol). Afterstirring at RT for 20 h, the reaction was quenched with hydrochloricacid (1 M, 100 mL). The aqueous layer was extracted with ethyl acetate(2×50 mL). The organics were combined, washed with saturated sodiumbicarbonate, dried over sodium sulfate, filtered and the volatiles wereremoved in vacuo. The residue was purified by silica gel chromatographyusing a 50 g Isolute cartridge eluted with a continuous gradient ofiso-hexanes/ethyl acetate 1:0 to 1:4 to afford the title compound(1.6735 g, 84%) as a brown foam.

A solution of 35e (534.7 mg, 1.013 mmol) in tetrahydrofuran (20 mL) wassubsequently treated with zinc powder (1.457 g, 22.286 mmol) and asolution of ammonium acetate (1.171 g, 15.195 mmol) in water (5 mL).After stirring at RT for 24 h, the mixture was filtered through Celite.The solid was rinsed with saturated potassium bisulfate and ethylacetate. The pH of the filtrate was adjusted with 2 M hydrochloric acidthen the aqueous layer was extracted with ethyl acetate (2×50 mL). Theorganics were combined, dried over sodium sulfate, filtered and thevolatiles were removed in vacuo. Residual acetic acid was azeotroped offwith toluene (3×) to provide the corresponding acid as a white solid. Asolution of the crude acid, (R)-1-(5-allyloxy-2-fluoro-phenyl)-ethanol(179.4 mg, 0.914 mmol) and 4-dimethylaminopyridine (123.7 mg, 1.013mmol) in dichloromethane (15 mL) was treated withN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (310.8 mg,1.621 mmol). After stirring at RT for 20 h, the reaction was quenched at0° C. with hydrochloric acid (2 M, 15 mL). The aqueous layer wasextracted with ethyl acetate (2×). The organics were combined, driedover sodium sulfate, filtered and the volatiles were removed in vacuo.The residue was purified by silica gel chromatography using a 50 gIsolute cartridge eluted with a continuous gradient of iso-hexanes/ethylacetate 1:0 to 1:3 to afford the title compound (310.5 mg, 59% over 2steps) as a white solid.

A solution of 35f (310.5 mg, 0.540 mmol) in dichloroethane (180 mL) wastreated with Hoveyda-Grubbs 2^(nd) generation catalyst (67.7 mg, 0.108mmol). After stirring at reflux for 2 h, the reaction was cooled to RTand the volatiles were removed in vacuo. The residue was purified bysilica gel chromatography using a 50 g Isolute cartridge eluted with acontinuous gradient of iso-hexanes/ethyl acetate 1:0 to 0:1 then using a25 g Isolute cartridge eluted by gravity with a continuous gradient ofiso-hexanes/ethyl acetate 1:0 to 0:1 to afford the title compound (109.9mg) in a mixture. Purification by preparative TLC eluted withiso-hexanes/acetone 3:1 (3 elutions) followed by silica gelchromatography using a 25 g Isolute cartridge eluted with a continuousgradient of iso-hexanes/acetone 1:0 to 3:2 provided the title compound(58.7 mg, 20%) as a white solid. ¹H NMR (300 MHz, CD₃CN) δ0.85-0.97 (m,6H), 1.17-1.22 (m, 3H), 1.28-1.39 (m, 4H), 1.44-1.63 (m, 6H), 1.70-1.82(m, 1H), 1.83-1.92 (m, 1H), 2.30-2.45 (m, 3H), 3.62-3.73 (m, 2H), 3.89(app t, J=8.5 Hz, 1H), 4.28 (d, J=8.0 Hz, 1H), 4.40-4.60 (m, 2H), 5.32(app pentet, J=6.9 Hz, 1H), 5.62-5.74 (m, 1H), 5.77-5.89 (m, 1H), 6.02(q, J=6.7 Hz, 1H), 6.40 (d, J=7.8 Hz, 1H), 6.76-6.85 (m, 2H), 7.01 (appt, J=9.4 Hz, 1H), 7.19 (br s, 1H). LCMS (m/z) 547.2 [M+H], Tr=2.39 min.

Example 36

To a solution of Compound 35 (44.0 mg, 0.080 mmol) in ethyl acetate (5mL) was added palladium on carbon (10%, 5 mg). The atmosphere was purgedof oxygen. After stirring at RT under an atmosphere of hydrogen for 2.5h, the volatiles were removed in vacuo and the residue was purified bysilica gel chromatography using a 20 g Isolute cartridge eluted with acontinuous gradient of iso-hexanes/acetone 1:0 to 3:2 followed bypreparative TLC eluted with iso-hexanes/acetone 3:2 to afford the finalcompound (19.0 mg, 43%) as a white solid. ¹H NMR (300 MHz, CDCl₃)δ0.85-0.99 (m, 8H), 1.14-1.22 (m, 4H), 1.26-1.42 (m, 4H), 1.48 (d, J=6.7Hz, 1H), 1.52-1.84 (m, 8H), 2.28-2.42 (m, 1H), 3.61-3.72 (m, 1H),3.75-4.10 (m, 4H), 4.25 (d, J=9.4 Hz, 1H), 5.26 (app pentet, J=7.1 Hz,1H), 6.02 (q, J=6.7 Hz, 1H), 6.34 (d, J=8.5 Hz, 1H), 6.75-6.86 (m, 2H),6.95-7.12 (m, 2H). LCMS (m/z) 549.3 [M+H], Tr=2.54 min.

Example 37:(E)-(2R,5S,11S,14S,17R,18R)-2,14-Diisopropyl-18-methoxy-11,17-dimethyl-22-oxa-3,9,12,15,28-pentaaza-tricyclo[21.3.1.1*5,9]octacosa-1(27),19,23,25-tetraene-4,10,13,16-tetraone

37a was prepared in the same manner as 35b using 3-hydroxybenzaldehydeinstead of 2-fluoro-5-hydroxy-N-methoxy-N-methyl-benzamide in 78% yield.

A cooled (0° C.) suspension of 37a (1.36 g, 8.434 mmol) and magnesiumsulfate (5 g) in dichloromethane (40 mL) was treated with D-valinol(870.0 mg, 8.434 mmol). After stirring at 0° C. to RT for 23 h, themixture was filtered and the volatiles were removed in vacuo. Theresidue was dissolved in anhydrous dichloromethane (40 mL) and wassubsequently treated with triethylamine (1.3 mL, 9.277 mmol) and asolution of trimethylsilyl chloride (9.3 mL, 9.277 mmol, 1 M indichloromethane). After stirring at RT for 24 h, the volatiles wereremoved in vacuo and the residue was triturated with diethylether/iso-hexane (100 mL, 1:1). The white solid was filtered off and thefiltrate was evaporated to dryness to provide the intermediate imine. Ina cooled (−40° C.) 3-neck round-bottom flask, equipped with a nitrogenline and an addition funnel was introduced anhydrous diethyl ether (25mL) and a solution of iso-propyllithium (29 mL, 20.242 mmol, 0.7 M inpentane). To this mixture, a solution of the imine in anhydrous diethylether (25 mL) was added dropwise over 15 min. After stirring at −40° C.for 2.5 h, the reaction was quenched with hydrochloric acid (2 M, 50 mL)and the mixture was allowed to warm to RT. The acidic aqueous layer wasbasified with NaOH pellets at 0° C. and then extracted with diethylether (2×). The organics were combined, dried over sodium sulfate,filtered and the volatiles were removed in vacuo. The residue waspurified by silica gel chromatography using a 50 g Isolute cartridgeeluted with a continuous gradient of iso-hexanes/ethyl acetate 1:0 to1:1 to afford the title compound (470.1 mg, 19% over 3 steps) as acolorless oil.

A solution of 37b (470.1 mg, 1.613 mmol) in methanol (10 mL) and aqueousmethylamine (3 mL, 40 wt % in water) was treated with periodic acid(1.213 g, 5.323 mmol). After stirring for 24 h at RT more aqueousmethylamine (4 mL, 40 wt % in water) and periodic acid (1.213 g, 5.323mmol) were added. After stirring for 17 h at RT the mixture was filteredover Celite and the solid rinsed with methanol. The volatiles wereremoved in vacuo and the residue was partitioned between water anddiethyl ether. The aqueous layer was extracted with diethyl ether, theorganics were combined, dried over sodium sulfate, filtered and thevolatiles were removed in vacuo. The residue was purified by silica gelchromatography using a 25 g Isolute cartridge eluted with ethyl acetateto afford the title compound (191.8 mg, 58%) as a light yellow oil.

A cooled (0° C.) solution of(S)-1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid (324.4 mg, 0.810 mmol), 37c (166.3 mg, 0.810 mmol) andN,N-diisopropylethylamine (560 μL, 3.240 mmol) in acetonitrile (15 mL)was treated with 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methanaminium (431.2 mg, 1.134 mmol). Afterstirring at RT for 20 h, the reaction was quenched with hydrochloricacid (2 M, 25 mL) at 0° C. The aqueous layer was extracted with ethylacetate (2×). The organics were combined, dried over sodium sulfate,filtered and the volatiles were removed in vacuo. The residue waspurified by silica gel chromatography using a 50 g Isolute cartridgeeluted with a continuous gradient of iso-hexanes/ethyl acetate 1:0 to0:1 to afford the title compound (230.5 mg, 48%) as a solid.

Compound 37e was prepared in the same manner as1-{(S)-2-[(S)-2-((E)-(2R,3R)-3-methoxy-2-methyl-hex-4-enoylamino)-3-methyl-butyrylamino]-propionyl}-3-methyl-hexahydro-pyridazine-3-carboxylicacid methyl ester using 37d instead of1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-propionyl]-3-methyl-hexahydro-pyridazine-3-carboxylicacid methyl ester in 30% yield over 2 steps.

Compound 37 was prepared in the same manner as 25 using 37e instead of(S)-1-{(S)-2-[(S)-2-((2R,3R)-3-methoxy-2-methyl-hept-6-enoylamino)-3-methyl-butyrylamino]-propionyl}-hexahydro-pyridazine-3-carboxylicacid methyl-((R)-6-vinyl-indan-1-yl)-amide in 43% yield. ¹H NMR (300MHz, d₆-DMSO) δ0.74-0.91 (m, 12H), 1.15 (d, J=7.1 Hz, 3H), 1.28 (d,J=7.3 Hz, 3H), 1.40-1.64 (m, 3H), 1.71-1.88 (m, 3H), 1.98 (app sextet,J=6.7 Hz, 1H), 2.66-2.75 (m, 2H), 3.38-3.53 (m, 1H), 3.89 (dd, J=6.7,2.9 Hz, 1H), 4.10 (app t, J=8.9 Hz, 1H), 4.14-4.22 (m, 1H), 4.42 (qd,J=10.0, 4.2 Hz, 1H), 4.57 (app t, J=8.2 Hz, 1H), 4.76 (d, J=11.8 Hz,1H), 5.23 (app t, J=7.3 Hz, 1H), 5.69-5.90 (m, 2H), 6.77-6.90 (m, 3H),7.05 (d, J=9.4 Hz, 1H), 7.16-7.24 (m, 1H), 8.01 (d, J=8.5 Hz, 1H), 8.14(d, J=8.5 Hz, 1H). LCMS (m/z) 586.3 [M+H], Tr=2.34 min.

Example 38

To a mixture of (R)-1-(3-bromo-phenyl)-ethanol (201 mg, 1.00 mmol),4-(2-ethoxy-2-oxoethoxy)benzeneboronic acid (Acros Organics, 224 mg,1.00 mmol) in 1,2-dimethoxyethane (4 mL) were added potassium carbonate(276 mg, 2.00 mmol) and water (1 mL). The mixture was stirred at RT andtetrakis(triphenylphosphine) palladium(0) (58 mg, 0.05 mmol) was addedthen the reaction mixture was heated at 100° C. in a microwave reactorfor 30 min. The reaction mixture was then diluted with ethyl acetate andwater. The layers were separated and the organics washed with water andbrine, dried over magnesium sulfate, filtered and evaporated. Theresidue was purified by silica gel chromatography using a gradient ofiso-hexanes/ethyl acetate 9:1 to 7:3 to afford the title compound (230mg, 76%) as an oil.

A solution of 38a (230 mg, 0.77 mmol) in tetrahydrofuran (4 mL) wasstirred at 5° C. under nitrogen, a solution of lithium hydroxidemonohydrate (92 mg, 1.54 mmol) was added and the reaction mixture wasstirred at 5° C. for 2 h and then at RT overnight. The solution wasacidified with 2 M hydrochloric acid and extracted with ethyl acetate(2×). The organic extracts were combined, washed with water and brine,dried over magnesium sulfate and evaporated to afford the title compound(175 mg, 84%) as a white solid.

A solution of 1e (372 mg, 0.70 mmol) in dichloromethane (15 mL) wascooled in an ice-water bath under nitrogen. Trimethylsilyltrifluoromethanesulfonate (0.18 mL, 1.05 mmol) was added dropwise, andthe resulting solution was stirred for 1 hour. Cold saturated aqueoussodium hydrogen carbonate solution (15 mL) was added and the mixture wasstirred at 0° C. for 15 min. The organic layer was separated, washedwith brine, dried over magnesium sulfate and evaporated to afford(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (0.70 mmol) which was used withoutfurther purification. A solution of(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (0.70 mmol) in acetonitrile (15 mL) wasstirred at 0° C. under nitrogen. 38b (175 mg, 0.64 mmol) and1-hydroxybenzotriazole hydrate (123 mg, 0.64 mmol, wetted with not lessthan 20 wt. % water) were added, followed byN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (173 mg,0.90 mmol) and the reaction mixture was stirred at RT for 2 h. Thesolvent was evaporated. The residue was dissolved in ethyl acetate andthe solution was washed with water (3×) followed by brine, dried overmagnesium sulfate and evaporated. The residue was purified by silica gelchromatography using a gradient of iso-hexanes/ethyl acetate 1:2 to 0:1.The product was triturated with diethyl ether and dried to afford thetitle compound (367 mg, 83%) as a white solid.

A solution of 38c (343 mg, 0.50 mmol) in tetrahydrofuran (10 mL) wasstirred at RT under nitrogen. Zinc powder (715 mg, 11 mmol) was addedfollowed by a solution of ammonium acetate (578 mg, 7.50 mmol) in water(5 mL). The reaction mixture was stirred at RT under nitrogen for 70 h.The reaction mixture was filtered through Celite and the filter pad waswashed with ethyl acetate and 2 M aqueous hydrochloric acid. Thefiltrate was acidified to pH 2 with 2 M aqueous hydrochloric acid, solidsodium chloride was added to saturate the aqueous layer and the mixturewas extracted with ethyl acetate. The ethyl acetate extracts werecombined, washed with brine, dried over magnesium sulfate andevaporated. The residue was co-evaporated with toluene (3×) to affordthe title compound (237 mg, 86%) as a white powder.

A solution of 38d (100 mg, 0.18 mmol) in dichloromethane (180 mL) wasstirred at RT under nitrogen. 4-Dimethylaminopyridine (33 mg, 0.27 mmol)and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (104mg, 0.54 mmol) were added and the reaction mixture was stirred at RT for18 h. The solvent was evaporated. The residue was purified by silica gelchromatography using a gradient of iso-hexanes/ethyl acetate 1:2 to 0:1followed by silica gel chromatography using a gradient of ethyl acetateto ethyl acetate/acetone 4:1. The residue was co-evaporated withdichloromethane then triturated with diethyl ether to afford a solid.The solid was washed with ether and dried to afford the title compound(8 mg, 9%) as a white solid. ¹H NMR (300 MHz, CDCl₃) 0.95 (d, J=6.2 Hz,3H), 0.96 (d, J=6.5 Hz, 3H), 1.39 (d, J=7.1 Hz, 3H), 1.54-1.80 (m, 5H),1.82-2.10 (m, 3H), 2.52-2.63 (m, 1H), 3.29-3.62 (m, 2H), 4.07 (app t,J=9.6 Hz, 1H), 4.45 (br d, J=13.6 Hz, 1H), 4.67 (ABq, Δδ_(AB)=0.12,J_(AB)=16.0 Hz, 2H), 4.88-5.01 (m, 1H), 5.93-6.00 (m, 2H), 6.57 (d,J=10.3 Hz, 1H), 6.94 (d, J=8.5 Hz, 2H), 7.15-7.53 (m, 6H). LCMS (m/z)537.2 [M+H], Tr=2.34 min.

Example 39

A solution of 4-iodo phenol (2.2 g, 10.0 mmol), (R)-2-hydroxy-propionicacid methyl ester (0.95 mL, 10.0 mmol) and triphenylphosphine (2.62 g,10.0 mmol) was prepared in tetrahydrofuran (40 mL). Diisopropylazodicarboxylate (2.0 mL. 10 mmol) was added dropwise and the reactionwas stirred at −5° C. for 1 hour and then at RT for 2 h. Thetetrahydrofuran was evaporated and diethyl ether/iso-hexanes (1:10, 50mL) was added. The mixture was stirred at RT for 10 min where aprecipitate formed. The filtrate was decanted off and evaporated. Theresidue was purified by silica gel chromatography using a gradient ofiso-hexanes/ethyl acetate 20:1 to 10:1 to afford the title compound(2.02 g, 66%) as an oil.

(R)-1-(3-bromophenyl)-ethanol (1.0 g, 4.97 mmol), bis(pinacolato)diboron(1.39 g, 5.47 mmol),1,1′bis(diphenylphosphino)ferrocenedichloropalladium(II),dichloromethane adduct (203 mg, 0.249 mmol) and potassium acetate (976mg, 9.94 mmol) were dissolved in 1,4-dioxane (10 mL) and the reactionwas heated to reflux and left to stir over 3 days. The reaction wasallowed to cool to RT before being filtered through a pad of Hyflo. Thepad was then washed with ethyl acetate and the combined organics werethen concentrated and purified by silica gel chromatography using astepwise gradient of iso-hexanes/ethyl acetate 1:0 to 4:1 to afford thetitle compound (936 mg, 76%) as a pale yellow oil.

A solution of 39b (496 mg, 2.00 mmol) and 39a (612 mg, 2.00 mmol) in1,2-dimethoxyethane (4 mL) was stirred at RT under nitrogen. A solutionof 2 M aqueous sodium carbonate (4 mL) was added followed bytetrakis(triphenylphosphine) palladium(0) (116 mg, 0.1 mmol) and thereaction mixture was heated at 80° C. for 1 hour. The reaction mixturewas cooled to RT, water was added and the mixture was extracted withethyl acetate. The ethyl acetate extracts were combined, washed withbrine, dried over sodium sulfate and evaporated. The residue waspurified by silica gel chromatography using a gradient ofiso-hexanes/ethyl acetate 17:3 to 1:1 to afford the title compound (253mg, 42%) as a gum.

A solution of 39c (250 mg, 0.83 mmol) in tetrahydrofuran (4 mL) wasstirred at 5° C. under nitrogen. A solution of lithium hydroxidemonohydrate (42 mg, 1.00 mmol) in water (1 mL) was added and thereaction mixture was stirred at 5° C. for 1 hour. The reaction mixturewas acidified to pH 3 with 2 M aqueous hydrochloric acid and the mixtureextracted with ethyl acetate. The organic extracts were separated,washed with water and brine, dried over magnesium sulfate and evaporatedto afford the title compound (224 mg, 94%) as a white foam.

A solution of 1e (478 mg, 0.9 mmol) in dichloromethane (15 mL) wascooled in an ice-water bath under nitrogen. Trimethylsilyltrifluoromethanesulfonate (0.25 mL, 1.35 mmol) was added dropwise, andthe resulting solution was stirred for 1 h. Cold saturated aqueoussodium hydrogen carbonate solution (15 mL) was added and the mixture wasstirred at 0° C. for 15 min. The organic layer was separated, washedwith brine, dried over magnesium sulfate and evaporated to afford(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (0.9 mmol) which was used withoutfurther purification. A solution of(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (0.9 mmol) in acetonitrile (15 mL) wasstirred at 0° C. under nitrogen. 39d (224 mg, 0.78 mmol) and1-hydroxybenzotriazole hydrate (184 mg, 0.96 mmol, wetted with not lessthan 20 wt. % water) were added, followed byN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (225 mg,1.17 mmol) and the reaction mixture was stirred at RT for 2 h. Thesolvent was evaporated. The residue was dissolved in ethyl acetate andthe solution was washed with water (3×) followed by brine, dried overmagnesium sulfate and evaporated. The residue was purified by silica gelchromatography using a gradient of iso-hexanes/ethyl acetate 1:2 to 0:1to afford the title compound (490 mg, 90%) as a gum.

A solution of 39e (490 mg, 0.70 mmol) in tetrahydrofuran (15 mL) wasstirred at RT under nitrogen. Zinc powder (1.00 g, 15.40 mmol) was addedfollowed by a solution of ammonium acetate (810 mg, 10.50 mmol) in water(8 mL). The reaction mixture was stirred at RT under nitrogen for 24 h.The reaction mixture was filtered through Celite and the filter pad waswashed with ethyl acetate and 2 M aqueous hydrochloric acid. Thefiltrate was acidified to pH 2-3 with 2 M aqueous hydrochloric acid.Solid sodium chloride was added to saturate the aqueous layer and themixture was extracted with ethyl acetate. The ethyl acetate extractswere combined and washed with brine. The organic extracts were passedthrough a hydrophobic frit and the filtrate was evaporated. The residuewas co-evaporated with ethyl acetate (3×) then toluene (3×) to affordthe title compound (394 mg, 98%) as a white solid.

A solution of 39f (100 mg, 0.18 mmol) in dichloromethane (180 mL) wasstirred at RT under nitrogen. 4-Dimethylaminopyridine (44 mg, 0.36 mmol)and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (138mg, 0.72 mmol) were added and the reaction mixture was stirred at RT for4 h. The solvent was evaporated. Ethyl acetate was added to the residueand the mixture was washed with aqueous citric acid (pH 4) and brine.The organic layer was dried over magnesium sulfate and the solvent wasevaporated. The residue was purified by silica gel chromatography usinga gradient of iso-hexanes/ethyl acetate 3:7 to 0:1. The residue wastriturated with diethyl ether/iso-hexanes 1:1 to afford the titlecompound (10 mg, 10%) as a white solid. ¹H NMR (300 MHz, CD₃OD) 0.94 (d,J=6.7 Hz, 3H), 0.95 (d, J=6.9 Hz, 3H), 1.51 (d, J=7.1 Hz, 3H), 1.61 (d,J=6.5 Hz, 3H), 1.67 (d, J=6.9 Hz, 3H), 1.72-2.00 (m, 5H), 2.60-2.69 (m,1H), 3.54-3.63 (m, 1H), 4.13 (d, J=10.5 Hz, 1H), 4.32 (brd, J=12.9 Hz,1H), 4.57-4.66 (m, 2H), 4.78 (q, J=7.1 Hz, 1H), 5.92 (q, J=6.5 Hz, 1H),6.91 (d, J=8.9 Hz, 2H), 7.22 (d, J=7.6 Hz, 1H), 7.34-7.61 (m, 5H). LCMS(m/z) 551.2 [M+H], Tr=2.51 min.

Example 40

40a was prepared in the same manner as (S)-2-(4-iodo-phenoxy)-propionicacid methyl ester using (S)-2-hydroxy-propionic acid ethyl ester insteadof (R)-2-hydroxy-propionic acid methyl ester in 28% yield.

40b was prepared in the same manner as(S)-2-[3′-((R)-1-hydroxy-ethyl)-biphenyl-4-yloxy]-propionic acid methylester using 40a instead of (S)-2-(4-iodo-phenoxy)-propionic acid methylester in 54% yield.

40c was prepared in the same manner as(S)-2-[3′-((R)-1-hydroxy-ethyl)-biphenyl-4-yloxy]-propionic acid using40b instead of(S)-2-[3′-((R)-1-hydroxy-ethyl)-biphenyl-4-yloxy]-propionic acid methylester in 71% yield.

40d was prepared in the same manner as(S)-1-[(S)-2-((S)-2-{(S)-2-[3′-((R)-1-hydroxy-ethyl)-biphenyl-4-yloxy]-propionylamino}-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester using 40c instead of(S)-2-[3′-((R)-1-hydroxy-ethyl)-biphenyl-4-yloxy]-propionic acid in 71%yield.

40e was prepared in the same manner as((S)-1-[(S)-2-((S)-2-{(S)-2-[3′-((R)-1-hydroxy-ethyl)-biphenyl-4-yloxy]-propionylamino}-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid using 40d instead of(S)-1-[(S)-2-((S)-2-{(S)-2-[3′-((R)-1-hydroxy-ethyl)-biphenyl-4-yloxy]-propionylamino}-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester in 83% yield.

A solution of 40e (102 mg, 0.18 mmol) in dichloromethane (180 mL) wasstirred at RT under nitrogen. 4-Dimethylaminopyridine (44 mg, 0.36 mmol)and (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate (114 mg, 0.22 mmol) were added and the reactionmixture was stirred at RT for 4 h. The solvent was evaporated. Ethylacetate was added to the residue and the mixture was washed with aqueouscitric acid (pH 4) and brine. The organic layer was separated and driedover magnesium sulfate and evaporated. The residue was purified bysilica gel chromatography using a gradient of iso-hexanes/ethyl acetate3:7 to 0:1. The residue was purified by preparative reverse phase HPLCto afford the title compound (2.8 mg, 3%) as a white solid. ¹H NMR (300MHz, CD₃OD) 1.03 (d, J=6.9 Hz, 3H), 1.05 (d, J=6.9 Hz, 3H), 1.25 (d,J=7.1 Hz, 3H), 1.56 (d, J=6.5 Hz, 3H), 1.57 (d, J=6.3 Hz, 3H), 1.64-2.10(m, 5H), 2.75-2.83 (m, 1H), 3.60-3.66 (m, 1H), 4.08 (d, J=10.0 Hz, 1H),4.32 (br d, J=12.9 Hz, 1H), 4.74 (q, J=6.5 Hz, 1H), 5.22 (q, J=7.3 Hz,1H), 5.98 (q, J=6.5 Hz, 1H), 6.86 (d, J=8.5 Hz, 2H), 7.20-7.25 (m, 1H),7.39-7.51 (m, 5H). LCMS (m/z) 551.2 [M+H], Tr=2.27 min.

Example 41

Dichloro (p-cymene) ruthenium(II) dimer (31 mg, 0.05 mmol) and(1R,2R)-(−)-N-p-tosyl-1,2-diphenylethylenediamine (44 mg, 0.12 mmol) wassuspended in degassed water (20 mL) and the mixture was degassed withnitrogen for 10 min. The mixture was stirred at 70° C. under nitrogenfor 90 min. The resulting yellow solution was cooled to RT. A solutionof 1-(6-bromo-pyridin-2-yl)-ethanone (2.0 mg, 10 mmol) in degassedtetrahydrofuran (10 mL) and sodium formate (3.4 g, 50 mmol) was addedand the reaction mixture was degassed with nitrogen for 5 min. Thereaction mixture was vigorously stirred at 40° C. for 30 min. Thereaction mixture was cooled to RT and was extracted with ethyl acetate.The organic extracts were combined, washed with water and brine, driedover sodium sulfate, filtered and evaporated. The residue was purifiedby silica gel chromatography using a gradient of iso-hexanes/ethylacetate 3:1 to 0:1 to afford the title compound (1.78 g, 89%) as a brownoil.

To a mixture of 41a (101 mg, 0.50 mmol),4-(2-ethoxy-2-oxoethoxy)benzeneboronic acid (112 mg, 0.50 mmol) in1,2-dimethoxyethane (4 mL) was added a solution of potassium carbonate(138 mg, 1.00 mmol) in water (1 mL).Tetrakis(triphenylphosphine)palladium(0) (29 mg, 0.025 mmol) was addedand the reaction mixture was heated at 100° C. in a microwave reactorfor 30 min. The reaction mixture was then diluted with ethyl acetate andwater. 2 M Hydrochloric acid was added to adjust the pH of the reactionmixture to pH 3 and the volatiles was evaporated. Methanol was added tothe residue and the mixture was filtered through a hydrophobic frit. Thefiltrate was evaporated and the residue was dried in vacuum to affordthe title compound (143 mg, 93%) as a white solid.

A solution of 1e (240 mg, 0.45 mmol) in dichloromethane (15 mL) wascooled in an ice-water bath under nitrogen. Trimethylsilyltrifluoromethanesulfonate (0.12 mL, 0.68 mmol) was added dropwise, andthe resulting solution was stirred for 1 h. Cold saturated aqueoussodium hydrogen carbonate solution (15 mL) was added and the mixture wasstirred at 0° C. for 15 min. The organic layer was separated, washedwith brine, dried over magnesium sulfate and evaporated to afford(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (0.45 mmol) which was used withoutfurther purification. A mixture of(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (0.45 mmol), 41b (143 mg, 0.45 mmol)and N,N-diisopropylethylamine (0.16 mL, 0.9 mmol) in acetonitrile (15mL) was stirred at RT under nitrogen. 1-Hydroxybenzotriazole hydrate(108 mg, 0.56 mmol, wetted with not less than 20 wt. % water) andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (130 mg,0.675 mmol) was added and the reaction mixture was stirred at RT for 4h. N,N-Dimethylformamide (2 mL) was added and the reaction mixture wasstirred at RT for 22 h. AdditionalN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (87 mg,0.45 mmol) was added and the reaction mixture was stirred at RT for 6 h.The solvent was evaporated. The residue was suspended in a mixture ofethyl acetate and aqueous citric acid solution (pH 3) and the mixturewas extracted with ethyl acetate. The organic extracts were combined,washed with water followed by brine. The organic layer was filteredthrough a hydrophobic frit and the filtrate was evaporated. The residuewas purified by silica gel chromatography using ethyl acetate to affordthe title compound (71 mg, 23%) as a white solid.

A solution of 41c (69 mg, 0.10 mmol) in tetrahydrofuran (2 mL) wasstirred at 0° C. under nitrogen, a solution of lithium hydroxidemonohydrate (5 mg, 0.12 mmol) in water (0.5 mL) was added and thereaction mixture was stirred at 0° C. for 1 h. The solution wasacidified to pH 3 with 2 M hydrochloric acid and the solvent wasevaporated. The residue was co-evaporated with toluene (3×) to afford(S)-1-{(S)-2-[(S)-2-(2-{4-[6-((R)-1-hydroxy-ethyl)-pyridin-2-yl]-phenoxy}-acetylamino)-3-methyl-butyrylamino]-propionyl}-hexahydro-pyridazine-3-carboxylicacid (58 mg, 0.1 mmol) as a white solid which was used crude in the nextstep. A solution of(S)-1-{(S)-2-[(S)-2-(2-{4-[6-((R)-1-hydroxy-ethyl)-pyridin-2-yl]-phenoxy}-acetylamino)-3-methyl-butyrylamino]-propionyl}-hexahydro-pyridazine-3-carboxylicacid (56 mg, 0.1 mmol) in dichloromethane (100 mL) was stirred at RTunder nitrogen. 4-Dimethylaminopyridine (49 mg, 0.4 mmol) was added andthe reaction mixture was for stirred for 5 min. A solution of(7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate(78 mg, 0.15 mmol) in dichloromethane (20 mL) was added dropwise over 5min and the reaction mixture was stirred at RT for 2 h. The solvent wasevaporated and the residue was purified by silica gel chromatographyusing ethyl acetate. The residue was purified by preparative reversephase HPLC to afford the title compound (14 mg, 26%) as a white solid.¹H NMR (300 MHz, CD₃OD) 0.94 (d, J=6.7 Hz, 3H), 0.98 (d, J=6.5 Hz, 3H),1.36 (d, J=7.1 Hz, 3H), 1.68 (d, J=6.5 Hz, 3H), 1.72-2.05 (m, 5H),2.65-2.74 (m, 1H), 3.53-3.60 (m, 1H), 4.14 (d, J=10.7 Hz, 1H), 4.30-4.38(m, 1H), 4.66 (ABq, Δδ_(AB)=0.13, J_(AB)=15.8 Hz, 2H), 5.07 (q, J=7.1Hz, 1H), 5.87 (q, J=6.5 Hz, 1H), 6.89 (d, J=8.9 Hz, 2H), 7.26 (br d,J=7.1 Hz, 1H), 7.71-7.78 (m, 2H), 8.06 (d, J=8.9 Hz, 2H). LCMS (m/z)538.2 [M+H], Tr=2.07 min.

Example 42

42a was prepared in the same manner as(S)-2-[3′-((R)-1-hydroxy-ethyl)-biphenyl-4-yloxy]-propionic acid methylester using 3-(4-bromo-pyrazol-1-yl)-propionic acid methyl ester insteadof (S)-2-(4-iodo-phenoxy)-propionic acid methyl ester in 28% yield.

42b was prepared in the same manner as(S)-2-[3′-((R)-1-hydroxy-ethyl)-biphenyl-4-yloxy]-propionic acid using42a instead of(S)-2-[3′-((R)-1-hydroxy-ethyl)-biphenyl-4-yloxy]-propionic acid methylester in 96% yield.

42c was prepared in the same manner as compound(S)-1-[(S)-2-((S)-2-{(S)-2-[3′-((R)-1-hydroxy-ethyl)-biphenyl-4-yloxy]-propionylamino}-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester using 42b instead of(S)-2-[3′-((R)-1-hydroxy-ethyl)-biphenyl-4-yloxy]-propionic acid in 49%yield.

Compound 42 was prepared in the same manner as Compound 41 using 42cinstead of(S)-1-{(S)-2-[(S)-2-(2-{4-[6-((R)-1-hydroxy-ethyl)-pyridin-2-yl]-phenoxy}-acetylamino)-3-methyl-butyrylamino]-propionyl}-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester in 8% yield. ¹H NMR (300 MHz, CD₃OD)0.94 (d, J=6.7 Hz, 3H), 0.95 (d, J=6.5 Hz, 3H), 1.37 (d, J=7.1 Hz, 3H),1.62 (d, J=6.5 Hz, 3H), 1.65-2.05 (m, 5H), 2.61-2.69 (m, 1H), 2.88-3.10(m, 2H), 3.58-3.63 (m, 1H), 3.90-4.00 (m, 1H), 4.01 (d, J=8.5 Hz, 1H),4.38-4.44 (m, 1H), 4.58-4.67 (m, 1H), 5.31 (q, J=7.1 Hz, 1H), 5.91 (q,J=6.5 Hz, 1H), 7.18 (d, J=7.6 Hz, 1H), 7.31 (app t, J=7.6 Hz, 1H), 7.47(d, J=7.6 Hz, 1H), 7.58 (s, 1H), 7.77 (s, 1H), 7.95 (s, 1H). LCMS (m/z)525.2 [M+H], Tr=4.51 min.

Example 43

Potassium carbonate (663 mg, 4.80 mmol) and tetrakis(triphenylphosphine)palladium (0) (139 mg, 0.12 mmol) were added to asolution of (R)-1-(3-bromophenyl)-ethanol (483 mg, 2.40 mmol) and4-(2-methoxy carbonylethyl) benzeneboronic acid (500 mg, 2.40 mmol) in1,2-dimethoxyethane (5 mL) in a 5 mL microwave vessel. The vessel wassealed before being heated in the microwave for 20 min, using fixed holdtime, on high absorption at 100° C. The reaction mixture was filteredthrough a pad of Hyflo and the pad was washed with ethyl acetate. Thecombined organics were then concentrated and the resultant brown oil waspurified by silica gel chromatography using a stepwise gradient ofiso-hexanes/ethyl acetate 1:0 to 3:2 to afford the title compound (416mg, 61%) as a yellow oil.

43a (416 mg, 1.46 mmol) was dissolved in a mixture of tetrahydrofuran (8mL) and water (2 mL) and the solution was cooled using an ice bath.Lithium hydroxide monohydrate (175 mg, 2.92 mmol) was added and thesolution was allowed to slowly warm to RT overnight. The solution wasacidified using 2 M hydrochloric acid and then extracted withdichloromethane (2×20 mL). The combined organics were then dried overanhydrous sodium sulfate, filtered and concentrated to afford the titlecompound (250 mg, 89%) as a white solid.

(S)-1-[(S)-2-((S)-2-Amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (669 mg, 1.55 mmol) and 43b (350 mg,1.29 mmol) were dissolved in anhydrous acetonitrile (10 mL) and cooledusing an ice bath. 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methanaminium (688 mg, 1.81 mmol) andN,N-diisopropylethylamine (899 μL, 5.16 mmol) were then added and thereaction was allowed to slowly warm to RT and left to stir overnight.The solvent was then removed and the residue was dissolved in ethylacetate. The solution was then washed with water (3×20 mL) and brine (20mL), dried over anhydrous sodium sulfate, filtered and concentrated andthe resultant brown oil was purified by silica gel chromatography usinga stepwise gradient of iso-hexanes/ethyl acetate 1:4 to 0:1 to affordthe title compound (314 mg, 36%) as a white solid.

43c (314 mg, 0.459 mmol) was dissolved in tetrahydrofuran (10 mL). Zincpowder (300 mg, 4.59 mmol) was added followed by ammonium acetate (1 Min water, 3.21 mL, 3.21 mmol). The reaction was then left to stirovernight. The reaction mixture was then filtered through a pad ofHyflo. The pad was then washed with potassium hydrogen sulfate solutionand ethyl acetate. The biphasic mixture was further acidified using 2 Mhydrochloric acid until the solution was approximately pH 1. The phaseswere separated and the aqueous phase was extracted with ethyl acetate.The combined organic phases were dried over anhydrous sodium sulfate,filtered and concentrated to afford the title compound (212 mg, 84%) asa yellow solid.

43d (100 mg, 0.181 mmol) was dissolved in dichloromethane (181 mL),under an atmosphere of nitrogen.N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (142 mg,0.724 mmol) and 4-dimethylaminopyridine (44 mg, 0.362 mmol) were addedand the reaction was left to stir overnight. The solvent was removed andthe residue was purified by silica gel chromatography using 100% ethylacetate to afford the title compound (52 mg, 54%) as a white solid. ¹HNMR (300 MHz, CDCl₃) δ0.92 (d, J=6.7 Hz, 6H), 1.24-1.29 (m, 3H),1.60-1.68 (m, 5H), 1.85-1.98 (m, 2H), 2.37-2.49 (m, 1H), 2.58-2.78 (m,1H), 2.83-2.93 (m, 1H), 3.16-3.28 (m, 1H), 3.45 (m, 3H), 4.03-4.11 (m,1H), 4.45-4.54 (m, 1H), 5.06-5.16 (m, 1H), 5.92-6.00 (m, 1H), 6.25 (d,J=8.5 Hz, 1H), 7.19 (s, 1H), 7.22 (s, 2H), 7.39 (app t, J=7.6 Hz, 1H),7.48-7.56 (m, 3H), 7.62 (br s, 1H). LCMS (m/z) 535.3 [M+H], Tr=2.43 min.

Example 44

Benzyl bromoacetate (2.07 mL, 13.1 mmol) and triphenylphosphine (3.63 g,13.8 mmol) were dissolved in toluene (60 mL) and left to stir over 3days. A white solid was collected by filtration and washed with diethylether. This afforded the title compound (5.86 g, 93%) as a white solid.

5-bromo-2-pyridine carboxaldehyde (125 mg, 0.666 mmol) and 44a (655 mg,1.33 mmol) were dissolved in dichloromethane (5 mL).1,1,3,3-Tetramethylguanidine (251 μL, 2 mmol) was then added and thereaction was left to stir for 3.5 h. The reaction was quenched withsaturated ammonium chloride solution and the phases were separated. Theorganic phase was then washed with brine, dried over anhydrous sodiumsulfate, filtered and concentrated. The residue was then purified bysilica gel chromatography using a stepwise gradient of iso-hexanes/ethylacetate 1:0 to 4:1 to afford the title compound (186 mg, 88%) as ayellow solid.

44b (186 mg, 0.585 mmol), (R)-1-[3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-ethanol (145 mg, 0.585 mmol), cesiumcarbonate (476 mg, 1.46 mmol) and tetrakis(triphenylphosphine) palladium(0) (66 mg, 0.0585 mmol) were placed in a microwave vessel.1,2-Dimethoxyethane (2 mL) and water (0.5 mL) were added and the vesselwas sealed.

The reaction was heated in the microwave for 30 min, using fixed holdtime, on high absorption at 150° C. The reaction mixture was poured ontowater and the resultant mixture was extracted with ethyl acetate. Theorganics were then dried over anhydrous sodium sulfate, filtered andconcentrated. The residue was then purified by silica gel chromatographyusing a stepwise gradient of iso-hexanes/ethyl acetate 1:0 to 1:1 toafford the title compound (150 mg, 77%) as an orange gum.

44c (150 mg, 0.417 mmol) was dissolved in ethanol (5 mL). Palladium(II)hydroxide (20% on carbon, wet, 40 mg) was then added followed byammonium formate (132 mg, 2.09 mmol). The reaction was heated to refluxand left to stir for 30 min. The reaction was allowed to cool to RT andwas then filtered through a pad of Hyflo. The pad was washed withethanol and the combined organics were concentrated to leave the titlecompound (105 mg, 93%) as an orange gum.

44d (105 mg, 0.387 mmol) and(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (200 mg, 0.464 mmol) were dissolved inacetonitrile (5 mL) and cooled using an ice bath.2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (206 mg, 0.542 mmol) andN,N-diisopropylethylamine (270 μL, 1.55 mmol) were then added and thereaction was allowed to slowly warm to RT and left to stir overnight.The solvent was then removed and the residue was dissolved in ethylacetate. The solution was then washed with water (3×20 mL) and brine (20mL), dried over anhydrous magnesium sulfate, filtered and concentratedand the residue was purified by silica gel chromatography using astepwise gradient of iso-hexanes/ethyl acetate/acetone 3:1:0 to 0:7:3 toafford a brown solid (110 mg). This was further purified by silica gelchromatography using a stepwise gradient of ethyl acetate/acetone 1:0 to4:1 to afford the title compound (100 mg, 38%) as a yellow solid.

44e (100 mg, 0.146 mmol) was dissolved in a mixture of tetrahydrofuran(3 mL) and water (1 mL) and cooled using an ice bath. Lithium hydroxidemonohydrate (6 mg, 0.153 mmol) was added and the reaction was stirredfor 30 min. The solution was acidified to pH 1 using 2 M hydrochloricacid and evaporated to dryness. The residue was then purified by C18chromatography using a stepwise gradient of acetonitrile/water 0:1 to1:4 to afford the title compound (25 mg, 31%) as a white solid.

44f (25 mg, 0.045 mmol) was dissolved in dichloromethane (45 mL).N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (35 mg,0.148 mmol) and 4-dimethylaminopyridine (11 mg, 0.009 mmol) were addedand the reaction was left to stir for 4 h. The solvent was removed andthe residue was purified by silica gel chromatography using a stepwisegradient of ethyl acetate/acetone 1:0 to 3:2 to leave a white solid (6mg). This was then eluted through an reverse phase HPLC system fittedwith a Phenomenex Gemini 10μ 110 A, 250×21.2 mm column using anisocratic 2:3 acetonitrile/water flow at 20 mL/min to afford the titlecompound (1.3 mg, 7%) as a white solid. ¹H NMR (300 MHz, CDCl₃)δ0.84-1.00 (m, 7H), 1.10 (d, J=6.9 Hz, 3H), 1.62-1.72 (m, 4H), 1.86-2.08(m, 3H), 2.54-2.71 (m, 2H), 2.90-3.14 (m, 2H), 3.28-3.54 (m, 3H), 3.98(app t, J=9.2 Hz, 1H), 4.43-4.52 (m, 1H), 5.07 (app t, J=8.0 Hz, 1H),5.92-6.00 (m, 1H), 6.10 (d, J=8.0 Hz, 1H), 6.37 (d, J=9.1 Hz, 1H), 7.20(d, J=8.0 Hz, 1H), 7.26-7.31 (m, 1H), 7.41-7.59 (m, 3H), 7.75 (dd,J=8.0, 2.2 Hz, 1H), 8.78 (d, J=2.4 Hz, 1H). LCMS (m/z) 536.0 [M+H],Tr=1.47 min.

Example 45

6-Bromo-3-pyridinecarboxaldehyde (500 mg, 2.69 mmol) andbenzyloxycarbonylmethyl-triphenyl-phosphonium bromide (2.64 g, 5.38mmol) were dissolved in dichloromethane (15 mL).1,1,3,3-Tetramethylguanidine (1.01 mL, 8.03 mmol) was then added and thereaction was left to stir for 3.5 h. The reaction was quenched withsaturated ammonium chloride solution and the phases were separated. Theorganic phase was then washed with brine, dried over anhydrous sodiumsulfate, filtered and concentrated. The residue was then purified bysilica gel chromatography using a stepwise gradient of iso-hexanes/ethylacetate 1:0 to 4:1 to afford the title compound (492 mg, 57%) as a whitesolid.

45a (382 mg, 1.54 mmol),(R)-1-[3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-ethanol(490 mg, 1.54 mmol), cesium carbonate (1.06 g, 3.85 mmol) andtetrakis(triphenylphosphine) palladium(0) (178 mg, 0.154 mmol) wereplaced in a microwave vessel. 1,2-Dimethoxyethane (4 mL) and water (1mL) were added and the vessel was sealed. The reaction was heated in themicrowave for 50 min, using fixed hold time, on high absorption at 150°C. The reaction mixture was poured onto water and the resultant mixturewas extracted with ethyl acetate. The organics were then dried overanhydrous sodium sulfate, filtered and concentrated. The residue wasthen purified by silica gel chromatography using a stepwise gradient ofiso-hexanes/ethyl acetate 1:0 to 1:1 to afford impure product. This wassubjected to a second round of purification using the same conditions toafford the title compound (195 mg, 35%) as a yellow solid.

45b (195 mg, 0.543 mmol) was dissolved in ethanol (5 mL). Palladium(II)hydroxide (20% on carbon, wet, 40 mg) was then added followed byammonium formate (172 mg, 2.72 mmol). The reaction was heated to refluxand left to stir for 30 min. The reaction was allowed to cool to RT andwas then filtered through a pad of Hyflo. The pad was washed withethanol and the combined organics were concentrated to leave the titlecompound (150 mg, 100%) as a pale yellow solid.

45c (150 mg, 0.553 mmol) and(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (287 mg, 0.664 mmol) were dissolved inacetonitrile (5 mL) and cooled using an ice bath.2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (294 mg, 0.774 mmol) andN,N-diisopropylethylamine (385 μL, 2.21 mmol) were then added and thereaction was allowed to slowly warm to RT and left to stir overnight.Ethyl acetate was then poured into the reaction mixture. The resultantsolution was then washed with water (3×20 mL) and brine (20 mL), driedover anhydrous sodium sulfate, filtered and concentrated and the residuewas purified by silica gel chromatography using a stepwise gradient ofiso-hexanes/ethyl acetate/acetone 3:1:0 to 0:7:3 to afford a solid (334mg). This was further purified by silica gel chromatography using astepwise gradient of ethyl acetate/acetone 1:0 to 4:1 to afford thetitle compound (167 mg, 44%) as a solid.

45d (160 mg, 0.234 mmol) was dissolved in a mixture of tetrahydrofuran(4 mL) and water (1 mL) and cooled using an ice bath. Lithium hydroxidemonohydrate (6 mg, 0.153 mmol) was added and the reaction was stirredfor 30 min. The solution was neutralized using 2 M hydrochloric acid andevaporated to dryness. The residue was then purified by C18chromatography using a stepwise gradient of acetonitrile/water 0:1 to1:4 to afford the title compound (21 mg, 16%) as a white solid.

45e (21 mg, 0.0379 mmol) was dissolved in dichloromethane (38 mL).N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (29 mg,0.152 mmol) and 4-dimethylaminopyridine (9 mg, 0.0758 mmol) were addedand the reaction was left to stir for 4 h. The solvent was removed andthe residue was purified by silica gel chromatography using 100% ethylacetate. The resultant material was then eluted through a reverse phaseHPLC system fitted with a Phenomenex Gemini 10μ 110 A, 250×21.2 mmcolumn using an isocratic 3:7 acetonitrile/water flow at 20 mL/min toafford the title compound (1 mg, 5%). ¹H NMR (300 MHz, CDCl₃) δ0.91 (d,J=6.5 Hz, 6H), 1.48 (d, J=7.1 Hz, 3H), 1.63-1.72 (m, 7H), 1.81-1.93 (m,2H), 2.00-2.10 (m, 1H), 2.25-2.38 (m, 1H), 2.54-2.67 (m, 1H), 3.15-3.28(m, 1H), 3.39-3.59 (m, 2H), 4.03 (app t, J=9.6 Hz, 1H), 4.43-4.54 (m,1H), 5.04-5.16 (m, 1H), 5.80 (d, J=9.8 Hz, 1H), 5.94 (q, J=6.7 Hz, 1H),7.22-7.27 (m, 1H), 7.43 (app t, J=7.8 Hz, 1H), 7.55-7.57 (m, 1H), 7.69(d, J=8.0 Hz, 1H), 7.92 (d, J=8.3 Hz, 1H), 8.19 (br s, 1H), 8.48 (d,J=2.2 Hz, 1H). LCMS (m/z) 536.2 [M+H], Tr=1.61 min.

Example 46

To Compound 21 (25 mg, 0.05 mmol) in ethyl acetate (5 mL) at RT wasadded 10% palladium on carbon (20 mg). The system was purged withhydrogen and stirred for 2 h. The reaction was filtered through Celiteand concentrated in vacuo. The residue was purified by preparative TLCusing ethyl acetate/acetone 5/1 to give the title compound (2.2 mg, 9%)as a white solid. ¹H NMR (300 MHz, CDCl₃) 0.96 (d, J=6.7 Hz, 3H), 0.98(d, J=6.7 Hz, 3H), 1.24 (d, J=7.1 Hz, 3H), 1.52 (d, J=6.9 Hz, 3H), 1.67(d, J=6.7 Hz, 3H), 1.55-2.14 (m, 10H), 2.53-2.80 (m, 2H), 2.92-3.01 (m,2H), 3.45-3.67 (m, 1H), 4.14-4.23 (m, 1H), 4.49-4.60 (m, 1H), 5.39-5.51(m, 1H), 5.96 (d, J=9.4 Hz, 1H), 6.06 (q, J=6.7 Hz, 1H), 6.43 (d, J=8.5Hz, 1H), 7.39 (dd, J=8.5, 1.1 Hz, 1H), 7.56 (s, 1H), 7.75 (s, 1H), 7.93(d, J=8.5 Hz, 1H), 9.16 (s, 1H). LCMS (m/z)=552.3 [M+H], Tr=1.25 min.

Example 47

To a solution of(S)-1-((S)-2-tert-butoxycarbonylamino-propionyl)-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (320 mg, 0.74 mmol) in dichloromethane(8 mL) was added trifluoroacetic acid (2 mL). The reaction was stirredat RT for 105 min and was then concentrated in vacuo. The resultingcrude product was dissolved in ethyl acetate (75 mL) and was washed withsaturated aqueous sodium bicarbonate. The aqueous phase was extractedwith ethyl acetate (2×40 mL), and the organics were dried over anhydroussodium sulfate, filtered, and concentrated to a crude residue that wasused without further purification. The residue from the previous step,1-hydroxybenzotriazole (152 mg, 1.12 mmol) and(S)-2-tert-butoxycarbonylamino-3-hydroxy-propionic acid (154 mg, 0.75mmol) were dissolved in dichloromethane (6 mL).N,N-Diisopropylethylamine (190 mg, 1.5 mmol) was added, and theresulting solution was cooled in an ice water bath.N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (149 mg, 0.96 mmol) wasadded dropwise over 15 s. The reaction was stirred for 18 h, allowingthe ice bath to slowly expire. The reaction mixture was then dilutedwith ethyl acetate (50 mL) and was washed with saturated aqueous sodiumbicarbonate and brine. The organic phase was dried over anhydrous sodiumsulfate, filtered, and concentrated to afford a crude residue that waspurified by silica gel chromatography (75 to 100% ethyl acetate iniso-hexanes) to afford the title product (211 mg, 55%) as an oil.

To a solution of 47a (64 mg, 0.12 mmol) in dichloromethane (1 mL) wasadded 2,4,6-collidine (98 mg, 1.28 mmol). tert-Butyldimethylsilyltrifluoromethanesulfonate (98 mg, 0.37 mmol) was added dropwise over 20sec. The reaction mixture was stirred for 15 h and was quenched withsaturated aqueous sodium bicarbonate (1 mL). The mixture was furtherdiluted with ethyl acetate, water, and 0.1 N aqueous hydrochloric acidto afford an acidic aqueous layer. The phases were separated, and theorganics were dried over sodium sulfate, filtered, and concentrated invacuo to afford a crude residue that was used without furtherpurification. The crude product was dissolved in tetrahydrofuran (3 mL).Acetic acid (115 mg, 1.9 mmol) was added in one portion and theresulting solution was stirred for 3.25 h. The reaction was then dilutedwith ethyl acetate (25 mL) and washed with saturated aqueous sodiumbicarbonate (25 mL). The phases were separated, and the aqueous phasewas extracted with ethyl acetate (25 mL). The combined organic phaseswere washed with brine (25 mL) and dried over anhydrous sodium sulfate.The mixture was filtered, and the filtrate was concentrated in vacuo toafford crude amine which was used without further purification. Thecrude amine was dissolved in N,N-dimethylformamide (1.5 mL).2,4,6-Collidine (31 mg, 0.26 mmol) and trans-3-pentenoic acid (15.3 mg,0.152 mmol) were added, and the resulting solution was cooled in an icewater bath.(1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbeniumhexafluorophosphate (72.4 mg, 0.169 mmol) was added in one portion, andthe reaction was stirred for 30 min. The reaction was then removed fromthe cold bath and warmed to ambient temperature. After 15 h, thereaction was diluted with ethyl acetate (35 mL), saturated aqueoussodium bicarbonate (20 mL), and brine (5 mL). The phases were separated,and the organic layer was washed with 0.1 N aqueous hydrochloric acid(25 mL) and then brine (5 mL). The acidic aqueous layer was extractedwith ethyl acetate (25 mL), and the combined organic phases were thendried over anhydrous sodium sulfate, filtered, and concentrated. Theresulting crude residue was purified by silica gel chromatography (50 to80% ethyl acetate in iso-hexanes) to afford the title compound (46.9 mg,62% over 3 steps) as a white foam.

To a solution of 47b (46.9 mg, 0.0761 mmol) in tetrahydrofuran (1.5 mL)was added water (0.30 mL), ammonium acetate (96 mg, 1.2 mmol), and zincpowder (109 mg, 1.7 mmol). The reaction mixture was stirred vigorouslyat RT for 17.5 h, at which time the temperature was increased to 35° C.After 25.5 h, additional zinc powder (60 mg, 0.92 mmol) was added andthe reaction temperature was increased to 45° C. After 39.5 h, thereaction mixture was filtered through a pad of Celite washing with waterand ethyl acetate. The aqueous phase was acidified with 2 M aqueoushydrochloric acid (15 mL), and the phases were separated. The aqueousphase was extracted with ethyl acetate (2×20 mL), and the combinedorganic phases were dried over sodium sulfate, filtered, andconcentrated to afford a white solid (34.9 mg, 95%) that was usedwithout further purification. The crude acid (0.072 mmol) was dissolvedwith (R)-1-(3-vinyl-isoquinolin-6-yl)-ethanol (17.4 mg, 0.087 mmol) indichloromethane (1.0 mL). 4-Dimethylaminopyridine (13.3 mg, 0.109 mmol)was added followed by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (17 mg, 0.085 mmol). The reaction was stirred for 16.5 h,at which time it was loaded directly onto a silica gel column. Elutionwith 60 to 100% ethyl acetate in iso-hexanes provided the title compound(20 mg, 43%) as an amorphous residue.

A solution of 47c (19.6 mg, 0.0294 mmol) in toluene (9.4 mL) was spargedwith argon for 10 min with stirring. Hoveyda-Grubbs 2^(nd)generation—catalyst (2.8 mg, 0.0045 mmol) was then added as a solutionin degassed toluene (0.45 mL), and the resulting solution was heated to105° C. After 25 min, an additional portion of Hoveyda-Grubbs 2^(nd)generation catalyst (1.4 mg, 0.0022 mmol) was added. After 10 additionalmin, the reaction was cooled to ambient temperature and was concentratedin vacuo to −4.5 mL. The solution was loaded directly onto a silica gelcolumn which was eluted with 80 to 100% ethyl acetate in iso-hexanes toafford the title compound (6.9 mg, 38%) as an amorphous solid.

In a polypropylene vial, 47d (4.7 mg, 0.0075 mmol) was dissolved intetrahydrofuran (0.90 mL) under argon and the resulting solution wascooled in an ice water bath. HF.pyridine (0.10 mL) was added dropwise.After 10 min, the reaction was quenched by addition to a stirred mixtureof ethyl acetate (15 mL) and saturated aqueous sodium bicarbonate (15mL). The phases were separated, and the aqueous phase was extracted withethyl acetate (3×30 mL). The combined organic phases were dried oversodium sulfate, filtered and concentrated to afford a crude residue. Ina polypropylene vial under argon, the aforementioned residue wasdissolved in tetrahydrofuran (0.90 mL) and the resulting solution wascooled in an ice water bath. HF.pyridine (0.10 mL) was added dropwise,and the reaction mixture was removed from the cold bath. After 45 min,the reaction was worked up as described above to afford a crude residue.Purification by reverse-phase HPLC (5 to 100% acetonitrile in water,+0.1% trifluoroacetic acid) provided the title compound (1.6 mg, 34%) asan amorphous white solid. ¹H NMR (400 MHz, CD₃OD) 9.54 (s, 1H), 8.38 (d,J=8.7 Hz, 1H), 8.29 (s, 1H), 8.17 (s, 1H), 7.87 (d, J=8.5 Hz, 1H),6.90-6.75 (m, 2H), 6.18 (q, J=6.9 Hz, 1H), 5.84-5.74 (m, 1H), 4.71-4.64(m, 1H), 4.50-4.42 (m, 1H), 3.88-3.81 (m, 2H), 3.78 (dd, J=11.3, 6.4 Hz,1H), 3.52 (dd, J=12.9, 6.2 Hz, 1H), 3.52 (dd, J=12.9, 6.2 Hz, 1H),2.81-2.70 (m, 1H), 2.11-2.04 (m, 1H), 1.99-1.91 (m, 1H), 1.85-1.69 (m,2H), 1.73 (d, J=6.7 Hz, 3H), 1.60 (d, J=7.1 Hz, 3H). LCMS (m/z) 510.2[M+H], Tr=2.19 min.

Example 48

To a solution of(S)-1-((S)-2-tert-butoxycarbonylamino-propionyl)-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (320 mg, 0.74 mmol) in dichloromethane(8.8 mL) was added trifluoroacetic acid (2.2 mL). The reaction wasstirred at RT for 45 min and was concentrated in vacuo. The resultingcrude product was twice dissolved in and concentrated from anhydroustoluene (10 mL). The resulting crude residue was used without furtherpurification. The crude residue (ca. 0.74 mmol), 1-hydroxybenzotriazole(153.8 mg, 1.1 mmol), and(2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-butyric acid (161.2 mg,0.735 mmol) were dissolved in dichloromethane (4 mL).N,N-Diisopropylethylamine (192 mg, 1.5 mmol) was added, and theresulting solution was cooled in an ice water bath.N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (132 mg, 0.85 mmol) wasadded dropwise over 15 s. The reaction was removed from the cold bathand was stirred for 21.5 h. The reaction mixture was then diluted withethyl acetate (50 mL) and washed with half-saturated aqueous sodiumbicarbonate (30 mL). The organic phase was washed with brine, dried overanhydrous sodium sulfate, filtered, and concentrated to afford a cruderesidue that was purified by silica gel chromatography eluting with 65to 90% ethyl acetate in iso-hexanes. Impure fractions were repurified bysilica gel chromatography and collated with the first product to providethe title compound (230 mg, 58%).

To a solution of 48a (62.3 mg, 0.12 mmol) in dichloromethane (1 mL) wasadded 2,4,6-collidine (146 mg, 1.21 mmol). tert-Butyldimethylsilyltrifluoromethanesulfonate (94 mg, 0.36 mmol) was added dropwise over 20s. The reaction mixture was stirred for 15 h and was quenched withsaturated aqueous sodium bicarbonate (1 mL). The mixture was furtherdiluted with ethyl acetate (2 mL) and brine (1 mL). The phases wereseparated, and the aqueous phase was extracted with ethyl acetate (4×1.5mL). The combined organic phases were dried over sodium sulfate,filtered, and concentrated in vacuo to afford a crude residue that wasused without further purification. The crude product was dissolved intetrahydrofuran (3 mL). Acetic acid (104 mg, 1.6 mmol) was added in oneportion and the resulting solution was stirred for 3.5 h. The reactionwas then diluted with ethyl acetate (30 mL), saturated aqueous sodiumbicarbonate (20 mL) and brine (10 mL). The phases were separated, andthe aqueous phase was extracted with ethyl acetate (20 mL). The combinedorganic phases were dried over anhydrous sodium sulfate. The mixture wasfiltered, and the filtrate was concentrated in vacuo to afford crudeintermediate amine which was used without further purification. Thecrude amine was dissolved in N,N-dimethylformamide (1.5 mL).2,4,6-Collidine (28 mg, 0.23 mmol) and trans-3-pentenoic acid (14.3 mg,0.143 mmol) were added, and the resulting solution was cooled in an icewater bath.(1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbeniumhexafluorophosphate (69 mg, 0.16 mmol) was added in one portion, and thereaction was stirred for 30 min. The reaction was then removed from thecold bath and warmed to ambient temperature. After 15 h, the reactionwas diluted with ethyl acetate (25 mL), 0.1 N aqueous hydrochloric acid(30 mL), and brine (5 mL). The phases were separated, and the acidicaqueous layer was extracted with ethyl acetate (25 mL). The combinedorganic phases were washed with saturated aqueous sodium bicarbonate (25mL), with brine (5 mL), dried over anhydrous sodium sulfate, filtered,and concentrated. The resulting crude residue was purified by silica gelchromatography (50 to 75% ethyl acetate in iso-hexanes) to afford thetitle compound (74 mg, quantitative yield over 3 steps) as a white foam.

To a solution of 48b (74 mg, 0.117 mmol) in tetrahydrofuran (2.3 mL) wasadded water (0.47 mL), ammonium acetate (138 mg, 1.8 mmol), and zincpowder (164 mg, 2.5 mmol). The reaction mixture was stirred vigorouslyat RT for 17.5 h, at which time the temperature was increased to 35° C.After 25.5 h, additional zinc powder (85 mg, 1.3 mmol) was added and thereaction temperature was increased to 45° C. After 39.5 total hours, thereaction mixture was filtered through a pad of Celite, washing withwater and ethyl acetate. The aqueous phase was acidified with 2 Naqueous hydrochloric acid (15 mL), and the phases were separated. Theaqueous phase was extracted with ethyl acetate (2×20 mL), and thecombined organic phases were dried over anhydrous sodium sulfate,filtered, and concentrated to afford an amorphous white solid (50 mg,86%) that was used without further purification. The crude product (0.10mmol) was dissolved along with (R)-1-(3-vinyl-isoquinolin-6-yl)-ethanol(23.9 mg, 0.12 mmol) in dichloromethane (1.0 mL).4-Dimethylaminopyridine (15 mg, 0.12 mmol) was added followed byN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (17 mg,0.019 mmol). The reaction was stirred for 16 h, at which time it wasloaded directly onto a silica gel column. Elution with 50 to 85, andthen to 100% ethyl acetate in iso-hexanes provided the title compound(40 mg, 58%) as a solid.

A solution of 48c (26 mg, 0.037 mmol) in toluene (12 mL) was spargedwith argon for several min with stirring. Hoveyda-Grubbs 2^(nd)generation catalyst (3.4 mg, 0.0054 mmol) was then added as a solutionin degassed toluene (0.40 mL), and the resulting solution was heated to105° C. After 20 min, an additional portion of Hoveyda-Grubbs 2^(nd)generation catalyst (1.7 mg, 0.0027 mmol) was added as a solution intoluene (0.20 mL). After 40 additional min, the reaction was cooled toRT and was concentrated in vacuo to −3 mL. The solution was loadeddirectly onto a silica gel column, which was eluted with 70 to 100%ethyl acetate in iso-hexanes to afford the title compound (10 mg, 42%)as an amorphous solid.

In a polypropylene vial under argon atmosphere, 48d (10 mg, 0.016 mmol)was dissolved in tetrahydrofuran (900 μL). HF.pyridine (˜70% as HF, 100μL) was added dropwise via syringe, and the resulting solution wasstirred for 130 min. An additional aliquot of HF.pyridine (100 μL) wasthen added via syringe, and the resulting solution was stirred for anadditional 80 min. The reaction mixture was quenched by its carefuladdition to a stirred mixture of ethyl acetate (50 mL) and saturatedaqueous sodium bicarbonate (50 mL). The phases were separated, and theaqueous phase was extracted with ethyl acetate (2×30 mL). The combinedorganic phases were dried over anhydrous sodium sulfate, filtered, andconcentrated to afford a crude residue that was purified by reversephase HPLC (C18, 15 to 100% acetonitrile/water, 0.1% trifluoroaceticacid). Impure fractions from this run were re-purified in the samemanner to afford the title compound as the trifluoroacetic acid salt(5.0 mg, 49%) as an amorphous white solid following lyophilization. ¹HNMR (400 MHz, CD₃OD) δ9.55 (s, 1H), 8.38 (d, J=8.6 Hz, 1H), 8.22 (s,1H), 8.15 (s, 1H), 7.87 (dd, J=8.7, 1.4 Hz, 1H), 6.86-6.75 (m, 2H), 6.15(q, J=6.6 Hz, 1H), 5.80-5.70 (m, 1H), 4.47 (d, J=5.6 Hz, 1H), 4.45-4.38(m, 1H), 4.09-4.01 (m, 1H), 3.83-3.78 (m, 1H), 3.55 (dd, J=13.5, 5.6 Hz,1H), 3.11 (dd, J=13.2, 4.7 Hz, 1H), 2.78-2.67 (m, 1H), 2.09-1.99 (m,1H), 1.96-1.89 (m, 1H), 1.82-1.64 (m, 5H), 1.60-1.54 (m, 3H), 1.25 (d,J=6.3 Hz, 3H). LCMS (m/z) 524.6 [M+H], Tr=2.15 min.

Example 49

A solution of(S)-1-((S)-2-tert-butoxycarbonylamino-propionyl)-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (865 mg, 2 mmol) in dichloromethane (20mL) was cooled in an ice water bath. Trimethylsilyltrifluoromethanesulfonate (667 mg, 3 mmol) was added dropwise at 0° C.under argon, and the resulting solution was stirred at RT for 30 min.The reaction mixture was evaporated to dryness and the resulting cruderesidue was dissolved in anhydrous acetonitrile (25 mL) under argon. Thereaction mixture was stirred at 0° C.,(S)-4-azido-2-(tert-butoxycarbonylamino)butanoic acid dicyclohexylaminesalt (936 mg, 2.2 mmol) and N,N-diisopropylethylamine (1034 mg, 8 mmol)were added followed by 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methanaminium (1065 mg, 2.8 mmol). Thereaction mixture was stirred at RT for 48 h. The solvent was evaporated,the residue was dissolved in ethyl acetate (100 mL) and the solution waswashed with 20% water solution of citric acid (2×100 mL), water (100 mL)and brine (100 mL), dried over magnesium sulfate, filtered andevaporated. The residue was purified by silica gel chromatography(gradient from 0-40% ethyl acetate+methanol (4/1) in iso-hexanes) toafford the title compound (783 mg, 70%) as a white solid afterevaporation. R_(f)=0.40, iso-hexanes/ethyl acetate/methanol (6/4/1).

Under argon, (R)-1-(3-chloro-isoquinolin-6-yl)-ethanol (880 mg, 4.23mmol),(E)-2,2-dimethyl-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-but-3-enoicacid methyl ester (1.24 g, 4.88 mmol),PdCl₂(PCy₂(p-NMe₂Ph))₂(bis[(dicyclohexyl)(4-dimethylaminophenyl)phosphine]palladium(II)chloride)173 mg, 0.21 mmol) and potassium phosphate tribasic (2.64 g, 12.4 mmol)were dissolved in cyclopentyl methyl ether (11.9 mL) and water (5.1 mL).The resulting biphasic mixture was vigorously stirred at 90° C. for 3.5h, at which time the reaction was cooled to ambient temperature and wasdiluted with ethyl acetate (50 mL) and water (40 mL). The phases wereseparated, and the aqueous phase was extracted with ethyl acetate (2×50mL). The combined organic phases were dried over anhydrous magnesiumsulfate, filtered, and concentrated to afford a crude residue that waspurified by silica gel chromatography (25 to 60% ethyl acetate iniso-hexanes) to afford the title compound (1.07 g, 85%) as a yellow oil.

To a solution of 49b (600 mg, 2 mmol) in tetrahydrofuran (8 mL) wasadded methanol (4 mL), water (4 mL) and lithium hydroxide (96 mg, 4mmol). The resulting mixture was stirred at RT for 10 h and quenchedwith 1 M hydrochloric acid (4.2 mL, 4.2 mmol). The resulting solutionwas concentrated to a crude residue which was co-distilled twice withtetrahydrofuran (20 mL), twice with anhydrous acetonitrile (20 mL) andtwice with anhydrous toluene (20 mL). The resulting white solid wasdried under high vacuum overnight and it was used without furtherpurification (735 mg, quantitative yield).

A solution of 49a (169 mg, 0.302 mmol) in dichloromethane (10 mL) wascooled in an ice water bath. Trimethylsilyl trifluoromethanesulfonate(101 mg, 0.455 mmol) was added dropwise at 0° C. under argon, and theresulting solution was stirred at RT for 30 min. The reaction mixturewas evaporated to dryness and the resulting crude residue was dissolvedin anhydrous acetonitrile (20 mL) under argon. Reaction mixture wasstirred at 0° C.,(E)-4-[6-((R)-1-hydroxy-ethyl)-isoquinolin-3-yl]-2,2-dimethyl-but-3-enoicacid (123 mg, 0.333 mmol) and N,N-diisopropylethylamine (151 mg, 1.220mmol) were added followed by2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (161 mg, 0.423 mmol). The reactionmixture was stirred at RT for 2 days. The solvent was evaporated, theresidue was dissolved in ethyl acetate (50 mL) and the solution waswashed with 20% water solution of citric acid (2×50 mL), water (50 mL)and brine (50 mL), dried over magnesium sulfate, filtered andevaporated. The residue was purified by silica gel chromatography(gradient from 0-40% ethyl acetate and methanol mixture (4/1) iniso-hexanes) to afford the title compound (198 mg, 90%) as a white solidafter evaporation. R_(f)=0.18, iso-hexanes/ethyl acetate/methanol(6/4/1).

To a solution of 49d (170 mg, 0.23 mmol) in tetrahydrofuran (2 mL) wasadded methanol (1 mL), water (1 mL) and lithium hydroxide hydrate (7 mg,0.28 mmol). The mixture was stirred for 2 h at ambient temperature andwas quenched with aqueous 1 M hydrochloric acid (0.30 mL, 0.30 mmol).The resulting solution was concentrated to a crude residue which wasco-evaporated twice with tetrahydrofuran (5 mL), twice with anhydrousacetonitrile (5 mL) and twice with anhydrous toluene (5 mL). Theresulting white solid was dried under high vacuum overnight and usedwithout further purification (151 mg, quantitative yield). Intooven-dried, argon purged flask were placed 2-methyl-6-nitrobenzoicanhydride (317 mg, 0.92 mmol), 4-dimethylaminopyridine (337 mg, 2.76mmol) and anhydrous 1,2-dichloroethane (300 mL). The resulting solutionwas heated at 50° C., and the crude seco-acid was added dropwise bysyringe as a solution in dry N,N-dimethylformamide (5 mL) over 12 h. Anadditional portion of dry N,N-dimethylformamide (2×1 ml) was used tocomplete the quantitative transfer. After stirring for additional 2 h at50° C., the reaction mixture was transferred to a separatory funnel andwashed with water (200 mL, 10 mL of brine was added to support theseparation). The aqueous phase was extracted with dichloromethane (100mL). Combined organic extracts were washed with brine (100 mL) and driedover magnesium sulfate, filtered and concentrated under reducedpressure. The resulting residue was dissolved in ethyl acetate (250 mL)and was washed with water (300 mL, 10 mL of brine was added to supportthe separation). The aqueous phase was extracted with ethyl acetate (150mL). Combined organic extracts were washed with water (200 mL, 10 mL ofbrine was added to support the separation). Resulting aqueous phase wasextracted with ethyl acetate (150 mL). Combined organic extracts werewashed with brine (150 mL) and dried over magnesium sulfate, filteredand concentrated under reduced pressure. The residue was purified bysilica gel chromatography (gradient from 0-40% ethyl acetate andmethanol mixture (4/1) in iso-hexanes) to afford the title compound (76mg, 57%) as a white solid after evaporation. R_(f)=0.51, 10% methanol indichloromethane. ¹H NMR (400 MHz, CD₃OD) δ9.03 (s, 1H), 7.95 (d, J=8.5Hz, 1H), 7.81 (s, 1H), 7.49 (s, 1H), 7.46 (dd, J=8.5, 1.6 Hz, 1H), 6.49(d, J=16.1 Hz, 1H), 6.40 (d, J=16.1 Hz, 1H), 5.95 (q, J=6.6 Hz, 1H),5.51 (q, J=7.2 Hz, 1H), 4.67 (dd, J=8.6, 6.3 Hz, 1H), 4.29 (m, 1H), 3.68(dd, J=11.2, 2.7 Hz, 1H), 3.28 (td, J=6.8, 3.7 Hz, 2H), 2.67-2.56 (m,1H), 1.88 (m, 2H), 1.85-1.60 (m, 4H), 1.57 (d, J=6.7 Hz, 3H), 1.54 (d,J=7.3 Hz, 3H), 1.42 (s, 3H), 1.27 (s, 3H). LCMS (m/z) 557.3 ([M+H],Tr=3.15 min.

Example 50

Into an oven-dried, argon purged flask, Compound 49 (20 mg, 0.034 mmol),copper(I) iodide (1 mg, 0.005 mmol) and propiolic acid (5 mg, 0.070mmol) were added. The flask was sealed with septa and repurged withargon three times. Anhydrous N,N-dimethylformamide (5 mL) was added andthe reaction mixture was repurged with argon three times. This reactionmixture was heated at 70° C. for 2 days. After evaporation of thesolvent under reduced pressure, the crude residue was dissolved in ethylacetate (10 mL) and filtered through Celite and the filter pad waswashed with ethyl acetate (10 mL). After concentration under reducedpressure, the residue was purified by silica gel chromatography(gradient from 0-40% ethyl acetate and methanol mixture (4/1) iniso-hexanes) to afford the title compound (7 mg, 34%) as a white solid.R_(f)=0.36, 5% methanol in dichloromethane. ¹H NMR (400 MHz, CD₃OD):δ9.05 (s, 1H), 8.11 (br s, 1H), 8.00 (br s, 1H), 7.95 (d, J=8.8 Hz, 1H),7.82 (s, 1H), 7.66 (s, 1H), 7.48 (d, J=10.0 Hz, 1H), 6.50 (d, J=16.2 Hz,1H), 6.42 (d, J=16.2 Hz, 1H), 5.97 (m, 1H), 5.56 (m, 1H), 4.60 (m, 1H),4.29 (m, 1H), 3.66 (m, 1H), 3.21 (m, 2H), 2.69-2.47 (m, 1H), 1.89 (m,2H), 1.87-1.55 (m, 4H), 1.53 (d, J=6.7 Hz, 3H), 1.50 (d, J=7.1 Hz, 3H),1.40 (s, 3H), 1.28 (s, 3H). LCMS (m/z) 603.1 [M+H]′ Tr=2.62 min.

Example 51

A solution of(S)-1-((S)-2-tert-butoxycarbonylamino-propionyl)-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (865 mg, 2 mmol) in dichloromethane (20mL) was cooled in an ice water bath. Trimethylsilyltrifluoromethanesulfonate (667 mg, 3 mmol) was added dropwise at 0° C.under argon, and the resulting solution was stirred at RT for 30 min.The reaction mixture was evaporated to dryness and the resulting cruderesidue was dissolved in anhydrous acetonitrile (25 mL) under argon.Reaction mixture was stirred at 0° C.,(S)-2-(tert-butoxycarbonylamino)pent-4-ynoic acid (469 mg, 2.2 mmol,source: Matrix Scientific, Catalog Number 041479) andN,N-diisopropylethylamine (1034 mg, 8 mmol) were added followed by2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (1065 mg, 2.8 mmol). Reaction mixturewas stirred at RT for 48 h. The solvent was evaporated, the residue wasdissolved in ethyl acetate (100 mL) and the solution was washed with 20%water solution of citric acid (2×100 mL), water (100 mL) and brine (100mL), dried over magnesium sulfate, filtered and evaporated. The residuewas purified by silica gel chromatography (gradient from 0-40% ethylacetate and methanol (4/1) in iso-hexanes) to afford the title compound(738 mg, 70%) as a white solid after evaporation. R_(f)=0.30,iso-hexanes/ethyl acetate/methanol (6/4/1).

A solution of 51a (145 mg, 0.275 mmol) in dichloromethane (10 mL) wascooled in an ice water bath. Trimethylsilyl trifluoromethanesulfonate(92 mg, 0.414 mmol) was added dropwise at 0° C. under argon, and theresulting solution was stirred at RT for 30 min. The reaction mixturewas evaporated to dryness and the resulting crude residue was dissolvedin anhydrous acetonitrile (20 mL) under argon. The reaction mixture wasstirred at 0° C.,(E)-4-[6-((R)-1-hydroxy-ethyl)-isoquinolin-3-yl]-2,2-dimethyl-but-3-enoicacid (112 mg, 0.303 mmol) and N,N-diisopropylethylamine (137 mg, 1.101mmol) were added followed by2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (147 mg, 0.385 mmol). The reactionmixture was stirred at RT for 2 days. The solvent was evaporated, theresidue was dissolved in ethyl acetate (50 mL) and the solution waswashed with 20% water solution of citric acid (2×50 mL), water (50 mL)and brine (50 mL), dried over magnesium sulfate, filtered andevaporated. The residue was purified by silica gel chromatography(gradient from 0-40% ethyl acetate and methanol (4/1) in iso-hexanes) toafford the title compound (163 mg, 85%) as a white solid afterevaporation. R_(f)=0.29, iso-hexanes/ethyl acetate/methanol (6/4/1).

To a solution of 51b (125 mg, 0.18 mmol) in tetrahydrofuran (2 mL) wasadded methanol (1 mL), water (1 mL) and lithium hydroxide hydrate(5.2mg, 0.22 mmol). The mixture was stirred for 2 h at ambient temperatureand was quenched with aqueous 1 M hydrochloric acid (0.25 mL, 0.25mmol). The resulting solution was concentrated to a crude residue whichwas co-evaporated twice with tetrahydrofuran (5 mL), twice withanhydrous acetonitrile (5 mL) and twice with anhydrous toluene (5 mL).The resulting white solid was dried under high vacuum overnight and itwas used without further purification (113 mg, quantitative yield). Intoan oven-dried, argon purged flask were placed 2-methyl-6-nitrobenzoicanhydride (248 mg, 0.72 mmol), 4-dimethylaminopyridine (264 mg, 2.16mmol) and anhydrous 1,2-dichloroethane (200 mL). The resulting solutionwas heated at 50° C., and the crude seco-acid was added dropwise viasyringe as a solution in anhydrous N,N-dimethylformamide (5 mL) over 12h. An additional portion of anhydrous N,N-dimethylformamide (2×1 ml) wasused to complete the quantitative transfer. After stirring foradditional 2 h at 50° C., the reaction mixture was transferred to aseparatory funnel and washed with water (200 mL, 10 mL of brine wasadded to support the separation). The aqueous phase was extracted withdichloromethane (100 mL). Combined organic extracts were washed withbrine (100 mL) and dried over magnesium sulfate, filtered andconcentrated under reduced pressure. The resulting residue was dissolvedin ethyl acetate (250 mL) and was washed with water (300 mL, 10 mL ofbrine was added to support the separation). The aqueous phase wasextracted with ethyl acetate (150 mL). Combined organic extracts werewashed with water (200 mL, 10 mL of brine was added to support theseparation). Resulting aqueous phase was extracted with ethyl acetate(150 mL). Combined organic extracts were washed with brine (150 mL) anddried over magnesium sulfate, filtered and concentrated under reducedpressure. The residue was purified by silica gel chromatography(gradient from 0-40% ethyl acetate+methanol (4/1) in iso-hexanes) toafford the title compound (56 mg, 57%) as a white solid afterevaporation. R_(f)=0.53, 10% methanol in dichloromethane. ¹H NMR (400MHz, CD₃OD) δ9.03 (s, 1H), 7.94 (d, J=8.5 Hz, 1H), 7.79 (s, 1H), 7.54(s, 1H), 7.45 (d, J=8.5 Hz, 1H), 6.52 (d, J=16.1 Hz, 1H), 6.40 (d,J=16.1 Hz, 1H), 5.95 (q, J=6.6 Hz, 1H), 5.52 (q, J=7.2 Hz, 1H), 4.70(dd, J=7.7, 6.7 Hz, 1H), 4.30 (m, 1H), 4.00 (q, J=7.2 Hz, 1H), 3.72-3.64(m, 1H), 2.63 (m, 1H), 2.55-2.40 (m, 2H), 1.91 (s, 1H), 1.88 (m, 1H),1.80 (m, 1H), 1.64 (m, 1H), 1.57 (d, J=6.7 Hz, 3H), 1.53 (d, J=7.2 Hz,3H), 1.42 (s, 3H), 1.27 (s, 3H). LCMS (m/z) 546.2 [M+H]′ Tr=3.04 min.

Example 52

A solution of Compound 51 (10 mg, 0.018 mmol) in a mixture of ethylacetate (4 mL) and tetrahydrofuran (4 mL) containing 5% palladium oncalcium carbonate poisoned with lead —Lindlar catalyst (10 mg) washydrogenated at RT and pressure of hydrogen for 3 h. The reactionmixture was filtered through Celite and the filter pad was washed withtetrahydrofuran (10 mL). The filtrate was evaporated to afford the titlecompound (10 mg, quantitative yield) as a white solid. R_(f)=0.19,iso-hexanes/ethyl acetate/methanol (6/4/1). ¹H NMR (400 MHz, CD₃OD):δ9.04 (s, 1H), 7.95 (d, J=8.4 Hz, 1H), 7.82 (s, 1H), 7.54 (s, 1H), 7.46(d, J=8.3 Hz, 1H), 6.50 (d, J=16.1 Hz, 1H), 6.39 (d, J=16.1 Hz, 1H),5.95 (q, J=6.4 Hz, 1H), 5.76-5.62 (m, 1H), 5.57 (q, J=7.0 Hz, 1H), 5.02(d, J=17.0 Hz, 1H), 4.96 (d, J=10.1 Hz, 1H), 4.61-4.54 (m, 1H), 4.29 (m,1H), 3.68n (m, 1H), 2.61 (m, 1H), 2.42-2.34 (m, 1H), 2.30-2.20 (m, 1H),1.90 (m, 1H), 1.81 (m, 1H), 1.68-1.60 (m, 2H), 1.57 (d, J=6.6 Hz, 3H),1.53 (d, J=7.2 Hz, 3H), 1.39 (s, 3H), 1.26 (s, 3H). LCMS (m/z) 548.3[M+H]′ Tr=2.85 min.

Example 53

To a solution of(S)-1-((S)-2-tert-butoxycarbonylamino-propionyl)-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (300 mg, 0.69 mmol) in dichloromethane(1.84 mL) was slowly added trifluoroacetic acid (460 μL, 6.00 mmol) at0° C. under an argon atmosphere. After 3 h, the reaction mixture wasconcentrated under reduced pressure. The crude residue was dissolved inN,N-dimethylformamide (3.45 mL) and(S)-tert-Butoxycarbonylamino-cyclohexyl-acetic acid (195 mg, 0.760mmol), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (289 mg, 0.760 mmol), andN,N-diisopropylethylamine (180 μL, 1.04 mmol) were sequentially added at23° C. under an argon atmosphere. After 18 h, the reaction mixture wasdiluted with ethyl acetate (300 mL), and the resulting mixture waswashed with brine (4×100 mL), was dried over anhydrous sodium sulfate,and was concentrated under reduced pressure. The crude residue waspurified by silica gel chromatography (12 g Combiflash HP Gold Column,0-100% ethyl acetate/iso-hexanes gradient) to afford the title compound(380 mg, 96%) as a colorless oil.

To a solution of 53a (220 mg, 0.385 mmol) in dichloromethane (1.92 mL)was added trimethylsilyl trifluoromethanesulfonate (128 mg, 0.587 mmol)at 0° C. under an argon atmosphere. After 1.5 h, the reaction mixturewas concentrated under reduced pressure. The resulting residue wasdiluted with acetonitrile (1.92 mL) and2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (146 mg, 0.385 mmol),N,N-diisopropylethylamine (267 μL, 1.54 mmol), and (E)-pent-3-enoic acid(39.4 μL, 0.385 mmol) were sequentially added at 23° C. under an argonatmosphere. After 20 h, the reaction mixture was diluted withdichloromethane (40 mL) and the resulting mixture was washed withsaturated aqueous sodium bicarbonate solution (40 mL) and with brine(2×40 mL). The organic layer was separated, was dried over anhydroussodium sulfate, and was concentrated under reduced pressure. The cruderesidue was purified by silica gel flash column chromatography (12 gCombiflash HP Gold Column, 0-100% ethyl acetate/iso-hexanes gradient) toafford the title compound (180 mg, 84%) as a colorless oil. R_(f)=0.75(ethyl acetate) I₂/silica stain.

To a solution of 53b (180 mg, 0.320 mmol) in tetrahydrofuran (5.3 mL)was added zinc powder (418 mg, 6.40 mmol) followed by a solution ofammonium acetate (370 mg, 4.80 mmol) in water (3.5 mL) at 23° C. underan argon atmosphere. After 15 h, the reaction mixture was warmed to 45°C. After 2 h, the reaction mixture was allowed to cool to RT and wasfiltered through a pad of Celite washing with water (10 mL) and ethylacetate (10 mL). The filtrate layers were split and the aqueous layerwas diluted with brine (80 mL) and was acidified to pH 1 with 12 Naqueous hydrogen chloride solution. The aqueous layer was extracted withethyl acetate (3×100 mL), and the combined organic extracts were driedover anhydrous sodium sulfate, and were concentrated under reducedpressure. Residual acetic acid was removed azeotropically via additionof toluene (5 mL) followed by concentration under reduced pressure (3×)to afford the title compound (74.5 mg, 55%) as a white solid.

To a solution of 53c (74.5 mg, 0.176 mmol) and(R)-1-(3-vinyl-isoquinolin-6-yl)-ethanol (42.1 mg, 0.212 mmol) indichloromethane (3.5 mL) were addedN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (47.2 mg,0.246 mmol) and 4-dimethylaminopyridine (11 mg, 88 μmol) at 23° C. underan argon atmosphere. After 18 h, the reaction mixture was purifieddirectly by silica gel chromatography (12 g Combiflash HP Gold Column,0-100% ethyl acetate/iso-hexanes gradient) to afford the title compound(67.6 mg, 64%) as a white solid.

To a solution of 53d (25 mg, 41 μmol) in toluene (8.2 mL) was added theHoveyda-Grubbs 2^(nd) Generation catalyst (2.5 mg, 4.1 μmol) at 23° C.under an argon atmosphere, and the resulting mixture was heated to 110°C. After 2 h, the reaction mixture was quenched with ethyl vinyl ether(300 μL) and the resulting mixture was allowed to cool to 23° C. Thereaction mixture was concentrated under reduced pressure and the cruderesidue was purified by silica gel chromatography (12 g Combiflash HPGold Column, 0-100% ethyl acetate/iso-hexanes gradient). The fractionscontaining the desired product were combined and were repurified bypreparatory HPLC to afford the title compound (1.7 mg, 7%) as a whitepowder as a trifluoroacetic acid salt. R_(f)=0.40 (ethyl acetate) UV. ¹HNMR (400 MHz, CD₃OD) δ9.13 (s, 1H), 8.53 (br s, 1H), 8.07 (d, J=7.6 Hz,1H), 8.05 (d, J=8.5 Hz, 1H), 7.83 (s, 1H), 7.55 (d, J=8.3 Hz, 1H), 7.44(s, 1H), 6.62 (d, J=16.3 Hz, 1H), 6.55-6.45 (m, 1H), 6.04 (q, J=6.5 Hz,1H), 5.50 (q, J=6.9 Hz, 1H), 4.40 (d, J=14.1 Hz, 1H), 4.33 (d, J=9.7 Hz,1H), 3.78 (d, J=11.0 Hz, 1H), 3.36 (app dd, J=14.7, 6.7 Hz, 1H), 2.99(dd, J=14.5, 4.9 Hz, 1H), 2.90-2.66 (m, 4H), 2.02-1.83 (m, 4H),1.82-1.70 (m, 3H), 1.68 (d, J=6.7 Hz, 3H), 1.65 (d, J=7.3 Hz, 3H),1.33-1.16 (m, 4H), 1.16-0.94 (m, 2H). HPLC Tr=t_(R) (min), 3.091(Synergi 4u hydro-RP, 50×4.60 mm 4 micron column, 7 min, 2 ml/min,5-100% acetonitrile/water, 0.05% trifluoroacetic acid modifiergradient). LCMS (m/z) 562.3 [M+H], Tr=2.17 min.

Example 54

To a solution of(S)-1-((S)-2-tert-butoxycarbonylamino-propionyl)-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (300 mg, 0.69 mmol) in dichloromethane(1.84 mL) was slowly added trifluoroacetic acid (460 μL, 6.00 mmol) at0° C. under an argon atmosphere. After 2 h, the reaction mixture wasconcentrated under reduced pressure. The crude residue was dissolved inN,N-dimethylformamide (3.45 mL) and(S)-2-tert-Butoxycarbonylamino-3-phenyl-propionic acid (201 mg, 0.760mmol), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (289 mg, 0.760 mmol), andN,N-diisopropylethylamine (180 μL, 1.04 mmol) were sequentially added at23° C. under an argon atmosphere. After 22 h, the reaction mixture wasdiluted with ethyl acetate (300 mL), and the resulting mixture waswashed with brine (3×200 mL), was dried over anhydrous sodium sulfate,and was concentrated under reduced pressure. The crude residue waspurified by silica gel chromatography (24 g Combiflash HP Gold Column,0-100% ethyl acetate/iso-hexanes gradient) to afford the title compound(319 mg, 96%) as a colorless oil. R_(f)=0.75 (ethyl acetate) I₂/silicastain.

To a solution of 54a (414 mg, 0.716 mmol) in dichloromethane (3.58 mL)was added trimethylsilyl trifluoromethanesulfonate (238.7 mg, 1.07 mmol)at 0° C. under an argon atmosphere. After 1 h, the reaction mixture wasconcentrated under reduced pressure. The resulting residue was dilutedwith acetonitrile (3.58 mL) and2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (272 mg, 0.716 mmol),N,N-diisopropylethylamine (498 μL, 2.86 mmol), and (E)-pent-3-enoic acid(73.3 μL, 0.716 mmol) were sequentially added at 23° C. under an argonatmosphere. After 17 h, the reaction mixture was diluted withdichloromethane (50 mL) and the resulting mixture was washed withsaturated aqueous sodium bicarbonate solution (50 mL). The organic layerwas separated, was dried over anhydrous sodium sulfate, and wasconcentrated under reduced pressure. The crude residue was purified bysilica gel chromatography to afford the title compound (386 mg, 96%) aswhite solid.

To a solution of 54b (443 mg, 0.789 mmol) in tetrahydrofuran (13.2 mL)was added zinc powder (1.03 g, 15.8 mmol) followed by a solution ofammonium acetate (912 mg, 11.8 mmol) in water (8.77 mL) at 23° C. underan argon atmosphere. After 17 h, the reaction mixture was warmed to 45°C. After 2 h, the reaction mixture was allowed to cool to RT and wasfiltered through a pad of Celite washing with water (10 mL) and ethylacetate (10 mL). The filtrate layers were split and the aqueous layerwas diluted with brine (20 mL) and was acidified to pH 2 with 12 Naqueous hydrogen chloride solution. The aqueous layer was extracted withdichloromethane (3×100 mL), and the combined organic extracts were driedover anhydrous sodium sulfate, and were concentrated under reducedpressure. Residual acetic acid was removed azeotropically via additionof toluene (5 mL) followed by concentration under reduced pressure (3×)to afford the title compound (276.2 mg, 81%) as a white solid.

To a solution of 54c (275 mg, 0.640 mmol) and(R)-1-(3-vinyl-isoquinolin-6-yl)-ethanol (153 mg, 0.770 mmol) indichloromethane (3.2 mL) were addedN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (172 mg,0.90 mmol) and 4-dimethylaminopyridine (39 mg, 32 μmol) at 23° C. underan argon atmosphere. After 23 h, the reaction mixture was purifieddirectly by silica gel chromatography (24 g Combiflash HP Gold Column,0-100% ethyl acetate/iso-hexanes gradient) to afford the title compound(203 mg, 52%) as a white solid.

To a solution of 54d (170 mg, 278 μmol) in chlorobenzene (56 mL) wasadded the Hoveyda-Grubbs 2^(nd) Generation catalyst (8.7 mg, 14.0 μmol)at 23° C. under an argon atmosphere, and the resulting mixture washeated to 110° C. After 3 h, the reaction mixture was quenched withethyl vinyl ether (300 μL) and the resulting mixture was allowed to coolto 23° C. The reaction mixture was concentrated under reduced pressureand the crude residue was purified by silica gel chromatography (12 gCombiflash HP Gold Column, 0-100% ethyl acetate/iso-hexanes gradient) toafford the title compound (18.9 mg, 12%) as a tan solid. R_(f)=0.25(ethyl acetate) UV. ¹H NMR (400 MHz, CD₃OD) δ9.12 (s, 1H), 8.05 (d,J=8.7 Hz, 1H), 7.84 (s, 1H), 7.56 (d, J=8.5 Hz, 1H), 7.53 (s, 1H),7.32-7.12 (m, 5H), 6.66 (d, J=16.5 Hz, 1H), 6.48 (dt, J=12.8, 5.6 Hz,1H), 6.05 (q, J=6.4 Hz, 1H), 5.53 (q, J=6.7 Hz, 1H), 4.73 (d, J=12.2 Hz,1H), 4.43 (d, J=11.9 Hz, 1H), 3.80 (app t, J=10.2 Hz, 1H), 3.30-3.22 (m,1H), 3.09 (dd, J=14.5, 4.9 Hz, 1H), 2.96-2.69 (m, 3H), 2.04-1.87 (m,2H), 1.82-1.56 (m, 2H), 1.68 (d, J=6.5 Hz, 3H), 1.64 (d, J=6.9 Hz, 3H).HPLC Tr=3.060 min. LCMS (m/z) 570.5 [M+H], Tr=2.14 min.

Example 55

To a solution of(S)-1-((S)-2-tert-butoxycarbonylamino-propionyl)-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (500 mg, 0.1.16 mmol) indichloromethane (5.8 mL) was added trimethylsilyltrifluoromethanesulfonate (386 mg, 1.74 mmol) at 0° C. under an argonatmosphere. After 1 h, the reaction mixture was concentrated underreduced pressure. The resulting residue was diluted with acetonitrile(5.8 mL) and 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (485 mg, 1.28 mmol),N,N-diisopropylethylamine (302 μL, 1.74 mmol),N-tert-butoxycarbnyl-3-(3-pyridyl)-L-alanine (337 mg, 1.27 mmol) weresequentially added at 23° C. under an argon atmosphere. After 20 h, thereaction mixture was concentrated under reduced pressure, and the cruderesidue was purified by silica gel chromatography (40 g Combiflash HPGold Column, 0-100% ethyl acetate/iso-hexanes gradient) to afford thetitle compound (570 mg, 85%) as light yellow oil.

To a solution of 55a (570 mg, 0.984 mmol) in dichloromethane (5.8 mL)was added trimethylsilyl trifluoromethanesulfonate (386 mg, 1.74 mmol)at 0° C. under an argon atmosphere. After 1 h, the reaction mixture wasconcentrated under reduced pressure to afford the title compound (478mg) as light yellow oil which was used without further purification.

To a solution of 55b (120 mg, 0.250 mmol) in acetonitrile (1.25 mL) weresequentially added(E)-4-[6-((R)-1-hydroxy-ethyl)-isoquinolin-3-yl]-2,2-dimethyl-but-3-enoicacid (75 mg, 0.25 mmol),2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (95 mg, 0.25 mmol), andN,N-diisopropylethylamine (173 μL, 1.00 mmol) at 23° C. under an argonatmosphere. N,N-Dimethylformamide (100 μL) was then added to promotesolubility of the reagents. After 23 h, the reaction mixture was dilutedwith saturated aqueous sodium bicarbonate solution (25 mL) and brine (25mL) and the resulting mixture was extracted with dichloromethane (2×25mL). The combined organic extracts were dried over anhydrous sodiumsulfate and were concentrated under reduced pressure. The crude residuewas purified by silica gel chromatography to afford the title compound(76 mg, 41%) as a colorless solid.

To a solution of 55c (76 mg, 0.10 mmol) in tetrahydrofuran (0.3 mL) andwater (0.2 mL) was added lithium hydroxide hydrate (2.4 mg, 0.10 mmol)at 23° C. under an argon atmosphere. After 2 h, the reaction mixture wasconcentrated under reduced pressure to afford the title compound (61 mg,99%) as a white solid lithium carboxylate salt.

To a solution of 55d (61 mg, 0.10 mmol) in dichloromethane (50 mL) wereadded benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(208 mg, 400 μmol) and 4-dimethylaminopyridine (366 mg, 3.00 mmol) at23° C. under an argon atmosphere. After 17 h, the reaction mixture wasconcentrated under reduced pressure and the crude residue was purifiedby silica gel chromatography (4 g Combiflash HP Gold Column, 0-20%methanol/dichloromethane gradient) to afford the title compound (1.2 mg,2%) as a colorless solid. R_(f)=0.40 (10% methanol in dichloromethane)UV. ¹H NMR (400 MHz, CD₃OD) δ9.03 (s, 1H), 8.31 (d, J=1.6 Hz, 1H), 8.27(dd, J=4.9, 1.5 Hz, 1H), 7.95 (d, J=8.5 Hz, 1H), 7.82 (s, 1H), 7.64 (d,J=8.0 Hz, 1H), 7.53 (s, 1H), 7.46 (dd, J=8.5, 1.5 Hz, 1H), 7.24 (dd,J=7.9, 4.9 Hz, 1H), 6.49 (d, J=16.1 Hz, 1H), 6.37 (d, J=16.1 Hz, 1H),5.95 (q, J=6.5 Hz, 1H), 5.57 (q, J=7.3 Hz, 1H), 4.65 (d, J=12.0 Hz, 1H),4.32 (br d, J=12.1 Hz, 1H), 3.83 (d, J=11.8 Hz, 1H), 3.73-3.65 (m, 1H),3.00 (dd, J=14.2, 5.0 Hz, 1H), 2.83 (dd, J=13.9, 9.7 Hz, 1H), 2.64 (brt, J=11.2 Hz, 1H), 1.95-1.73 (m, 2H), 1.69-1.59 (m, 1H), 1.57 (d, J=6.7Hz, 3H), 1.54 (d, J=7.2 Hz, 3H), 1.33 (s, 3H), 1.16 (s, 3H). HPLCTr=4.491 min

Example 56

To a solution of(S)-1-((S)-2-tert-butoxycarbonylamino-propionyl)-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (791 mg, 1.82 mmol) in dichloromethane(10.0 mL) was slowly added trimethylsilyl trifluoromethanesulfonate (483μL, 2.73 mmol) at 0° C. under an argon atmosphere. After 45 min, thereaction mixture was concentrated under reduced pressure. The resultingresidue was diluted with dichloromethane (10.0 mL) and2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (761 mg, 2.00 mmol),N,N-diisopropylethylamine (1.26 mL, 7.28 mmol), and(S)-2-(tert-butoxycarbonylamino)-4-phenylbutanoic acid (Fluka, 560 mg,2.00 mmol) were sequentially added at 23° C. under an argon atmosphere.After 18 h, the reaction mixture was concentrated under reducedpressure. The residue was pre-absorbed on silica and purified by silicagel chromatography (40 g Isco Rf Gold Column, 0-100% ethylacetate/iso-hexanes gradient) to afford the title compound (799 mg, 74%)as a colorless oil.

To a solution of 56a (799 mg, 1.34 mmol) in dichloromethane (10.0 mL)was added trimethylsilyl trifluoromethanesulfonate (356 μL, 2.01 mmol)at 0° C. under an argon atmosphere. After 30 min, the reaction mixturewas concentrated under reduced pressure. The resulting residue wasdiluted with acetonitrile (6.0 mL) and2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (560 mg, 1.47 mmol),N,N-diisopropylethylamine (932 μL, 5.36 mmol), and (E)-pent-3-enoic acid(150 μL, 1.47 mmol) were sequentially added at 23° C. under an argonatmosphere. After 19 h, the reaction mixture was concentrated underreduced pressure. The residue was pre-absorbed on silica and purified bysilica gel chromatography to afford the title compound (598 mg, 78%) asa colorless oil.

To a solution of 56b (167 mg, 0.29 mmol) in tetrahydrofuran (3 mL),methanol (1 mL) and water (1 mL) was added lithium hydroxide hydrate(7.6 mg, 0.32 mmol) at 23° C. After 15 min, the reaction mixture wasconcentrated to dryness under reduced pressure. The residue wasdissolved in water (25 mL) and washed with ethyl acetate. The organiclayer was extracted with saturated aqueous sodium bicarbonate solution(25 mL). The combined aqueous layers were acidified with 1 M aqueoushydrochloric acid solution to pH ˜2 and extracted with ethyl acetate(3×25 mL), and the combined organic extracts were dried over anhydrousmagnesium sulfate, and were concentrated under reduced pressure toafford the title compound (129 mg, 100%) as a white solid.

To a solution of 56c (129 mg, 0.29 mmol) and(R)-1-(3-vinyl-isoquinolin-6-yl)-ethanol (64 mg, 0.31 mmol) indichloromethane (3.0 mL) was addedN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (89 mg,0.46 mmol) and 4-dimethylaminopyridine (18 mg, 0.14 mmol) at 23° C.under an argon atmosphere. After 16 h, the reaction mixture was purifieddirectly by silica gel chromatography to afford the title compound (87mg, 48%) as a white solid.

To a solution of 56d (84 mg, 0.13 mmol) in chlorobenezene (27 mL) wasadded the Hoveyda-Grubbs 2^(nd) Generation catalyst (8 mg, 13 μmol) at23° C. under an argon atmosphere, and the resulting mixture was heatedto 110° C. After 2 h, the Hoveyda-Grubbs 2^(nd) Generation catalyst (7.3mg, 11 μmol) was added under argon at 110° C. The Hoveyda-Grubbs 2^(nd)Generation Catalyst (5 mg, 8 μmol) was then added in the interval of 30min for three times at which point the reaction was complete. Thereaction mixture was quenched with ethyl vinyl ether (1.0 μL) and theresulting mixture was allowed to cool to 23° C. The reaction mixture wasconcentrated under reduced pressure and the crude residue was purifiedby silica gel chromatography (24 g Isco Rf Gold Column, 0-100% ethylacetate/iso-hexanes gradient for 10 min and then 100% ethyl acetate for25 min) to afford the title compound (9.1 mg, 12%) as a pale brownsolid. R_(f)=0.20 (ethyl acetate). ¹H NMR (400 MHz, CD₃OD) δ9.00 (s,1H), 7.93 (d, J=8.5 Hz, 1H), 7.71 (s, 1H), 7.45 (dd, J=8.5, 1.5 Hz, 1H),7.36 (s, 1H), 7.20-7.08 (m, 4H), 7.08-7.00 (m, 1H), 6.55 (d, J=15.9 Hz,1H), 6.40 (ddd, J=15.9, 7.0, 5.1 Hz, 1H), 5.95 (q, J=6.5 Hz, 1H), 5.40(q, J=7.2 Hz, 1H), 4.60 (d, J=12.1 Hz, 1H), 4.53 (dd, J=8.2, 6.9 Hz,1H), 4.30 (d, J=12.5 Hz, 1H), 3.70-3.63 (m, 1H), 3.31-3.23 (m, 1H), 2.93(ddd, J=14.0, 5.1, 1.6 Hz, 1H), 2.69-2.52 (m, 3H), 2.01-1.73 (m, 4H),1.71-1.54 (m, 2H), 1.59 (d, J=6.7 Hz, 3H), 1.46 (d, J=7.3 Hz, 3H). HPLCTr=5.108 min. LCMS (m/z) 584.3 [M+H], Tr=2.12 min.

Example 57

A solution of(S)-1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methylbutyrylamino)-propionyl]-hexahydropyridazine-3-carboxylicacid 2,2,2-trichloroethyl ester (1.61 g, 3.03 mmol) in dichloromethane(31 mL) was cooled in an ice water bath under argon. Trimethylsilyltrifluoromethanesulfonate (1.23 g, 5.5 mmol) was added dropwise, and theresulting solution was stirred for 2 h. The reaction was quenched withN,N-diisopropylethylamine (1.2 g, 9.2 mmol) and methanol (8.5 mL). Themixture was concentrated in vacuo and was redissolved and concentratedfrom toluene (2×25 mL). The resulting crude residue containing(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (1.05 g) was used without furtherpurification.

To a solution of(E)-4-[6-((R)-1-hydroxy-ethyl)-isoquinolin-3-yl]-2,2-dimethyl-but-3-enoicacid methyl ester (1.05 g, 3.51 mmol) in tetrahydrofuran (8 mL) wasadded methanol (4 mL), water (4 mL), and lithium hydroxide hydrate (297mg, 7.08 mmol). The resulting mixture was stirred for 6 h and wasquenched with 1 M aqueous hydrochloric acid (7.2 mL, 7.2 mmol). Theresulting solution was concentrated to a crude residue that wasredissolved and concentrated from anhydrous methanol (50 mL) followed bytoluene (50 mL). The resulting yellow solid (1.3 g, 100%)(E)-4-[6-((R)-1-hydroxy-ethyl)-isoquinolin-3-yl]-2,2-dimethyl-but-3-enoicacid was used without further purification. A portion of crude(E)-4-[6-((R)-1-hydroxy-ethyl)-isoquinolin-3-yl]-2,2-dimethyl-but-3-enoicacid (1.05 g, 2.84 mmol) was dissolved in N,N-dimethylformamide (12 mL)under argon. N,N-diisopropylethylamine (1.85 g, 14.3 mmol) was addedfollowed by 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (1.42 g, 3.73 mmol). The resultingmixture was stirred for 3 min, at which time crude 57a was added as asolution in N,N-dimethylformamide (8.5 mL), washing with additionalN,N-dimethylformamide (2×5 mL). The reaction was stirred for 40 min andwas then diluted with ethyl acetate (200 mL) and water (300 mL). Thephases were separated, and the aqueous phase was extracted with ethylacetate (200 mL). The combined organic phases were washed with water(150 mL), and the second aqueous layer was extracted with ethyl acetate(100 mL). The combined organics were dried over anhydrous sodiumsulfate, filtered, and concentrated. The crude residue was purified bysilica gel chromatography (35 to 60% acetone in iso-hexanes, continuousgradient) to afford the title compound (1.51 g, 71% over 2 steps) as acolorless oil. R_(F) 0.5 (50% acetone in iso-hexanes).

To a solution of 57b (1.50 g, 2.15 mmol) in tetrahydrofuran (20 mL) wasadded methanol (10 mL), water (10 mL), and lithium hydroxide hydrate(365 mg, 8.7 mmol). The mixture was stirred for 75 min at ambienttemperature and was then quenched with aqueous 1 M hydrochloric acid(8.8 mL, 8.8 mmol). The resulting solution was concentrated in vacuo,and the crude product was twice dissolved and concentrated from methanol(40 mL) and suspended and concentrated from acetonitrile (6×30 mL) toafford 1.71 g of a colorless solid that was used without furtherpurification. Under argon, 2-methyl-6-nitrobenzoic anhydride (1.85 g,5.37 mmol) and 4-dimethylaminopyridine (1.97 g, 16.1 mmol) weredissolved in 1,2-dichloroethane (700 mL). The resulting solution washeated to 50° C., and the crude seco-acid was added dropwise via syringeas a solution in N,N-dimethylformamide (22 mL) over 6 h. An additionalwash with N,N-dimethylformamide (1.5 mL) was then added in the samemanner over 15 min. After stirring an additional 1.25 h, the reactionmixture was concentrated to a final volume of 200 mL in vacuo. Thesolution was washed with water (250 mL), and the aqueous phase wasextracted with dichloromethane (150 mL). The organic phase was driedover anhydrous magnesium sulfate, filtered, and concentrated. Theresulting residue was dissolved in ethyl acetate (200 mL) and was washedwith water (150 mL). The aqueous phase was extracted with ethyl acetate(150 mL). The combined organics were washed with water (100 mL), and thesecond aqueous phase was extracted with ethyl acetate (100 mL). Thecombined organics were dried over anhydrous magnesium sulfate, filteredand concentrated. The crude residue was purified by silica gelchromatography (35 to 65% acetone in iso-hexanes continuous gradient) toafford 445 mg of the pure title compound as an amorphous white solidalong with 389 mg of impure product. The impure fractions were purifiedby recrystallization from acetone:iso-hexanes to afford an additional173.5 mg of pure title compound (total: 618.5 mg, 52%). R_(F) 0.5 (50%acetone in iso-hexanes). ¹H NMR (400 MHz, CD₃OD) δ9.13 (s, 1H), 8.04 (d,J=8.5 Hz, 1H), 7.89 (s, 1H), 7.55 (dd, J=8.5, 1.6 Hz, 1H), 7.53 (s, 1H),6.73-6.34 (m, 2H), 6.03 (q, J=6.6 Hz, 1H), 5.63 (q, J=7.2 Hz, 1H), 4.67(d, J=12.2 Hz, 1H), 4.47-4.21 (m, 2H), 3.81-3.73 (m, 1H), 2.76-2.64 (m,1H), 2.08-1.81 (m, 3H), 1.80-1.58 (m, 8H), 1.51 (s, 3H), 1.35 (s, 3H),0.98 (d, J=6.7 Hz, 3H), 0.93 (d, J=6.7 Hz, 3H). LCMS (m/z) 550.2 [M+H],Tr=2.74 min.

Example 58

A solution of N,N-diisopropylamine (2.51 g, 24.8 mmol) intetrahydrofuran (150 mL) under argon was cooled in an ice water bath. Asolution of n-butyllithium in hexanes (2.5 M, 9.7 mL, 24 mmol) was addeddropwise over 2 min, and the resulting solution was stirred for 15additional minutes. The solution was then cooled to −78° C. viaCO_(2(s)):acetone bath, and ethyl 1-acetylcyclopropanecarboxylate (3.47g, 22.2 mmol) was added dropwise over 2 min. The solution was stirredfor an additional 20 min, and N-Phenyl-bis(trifluoromethanesulfonimide)(8.4 g, 23.5 mmol) was added as a solution in tetrahydrofuran (24 mL)via canula over 5 min, washing with additional portions oftetrahydrofuran (2×5 mL). The resulting solution was removed from thecold bath. After an additional 30 min, the reaction mixture wasconcentrated in vacuo and was diluted with diethyl ether (200 mL). Theorganic phase was washed with 1 M aqueous sodium hydroxide (1×100 mL,1×30 mL), dried over anhydrous magnesium sulfate, filtered, andconcentrated to afford 6.6 g of a crude oil that was used withoutfurther purification. A solution of the crude material from the previousstep in anhydrous pyridine (11 mL) was heated to 90° C. After 16.5 h,the reaction mixture was diluted with diethyl ether (200 mL) and 3 Mhydrochloric acid (100 mL) and the phases were separated. The organicphase was washed with 3 M hydrochloric acid (50 mL) and 1 M sodiumhydroxide (50 mL), dried over magnesium sulfate, filtered, andconcentrated to afford 2.2 g of a crude liquid that was used withoutfurther purification. To an argon-flushed vessel containing zirconocenedichloride (410 mg, 1.6 mmol) was added a solution of the crude productfrom the previous step (2.2 g, ca. 16 mmol) and pinacolborane (3.1 g, 24mmol) in dichloromethane (8 mL). After 116 h, the stirred reactionmixture was diluted with ethyl acetate (50 mL) and was quenched bydropwise addition of water. The mixture was further diluted with water(50 mL), and the phases were separated. The aqueous phase was extractedwith ethyl acetate (2×50 mL). The combined organic phases were driedover magnesium sulfate, filtered, and concentrated to a crude residuethat was purified by silica gel chromatography (5 to 20% ethyl acetatein iso-hexanes, continuous gradient) to afford the title compound (1.26g, 21% over 3 steps) as an oil that crystallized on standing at −15° C.

To a round-bottomed flask was added(R)-1-(3-chloroisoquinolin-6-yl)ethanol (204 mg, 0.982 mmol), 58a (314mg, 1.18 mmol), PdCl₂(PCy₂(p-NMe₂Ph))₂(bis[(dicyclohexyl)(4-dimethylaminophenyl)phosphine] palladium(II)chloride) (40 mg, 0.049 mmol) and potassium phosphate tribasic (680 mg,3.2 mmol). The vessel was sealed with a septum cap and was flushed withargon. Cyclopentyl methyl ether (2.8 mL) and water (1.2 mL) were added,and the resulting biphasic mixture was vigorously stirred in an oil bathpre-heated to 90° C. After 6.75 h, the reaction was cooled to ambienttemperature and was diluted with ethyl acetate (50 mL) and water (40mL). The phases were separated, and the aqueous phase was extracted withethyl acetate (50 mL). The combined organic phases were dried overmagnesium sulfate, filtered, and concentrated to afford a crude residuethat was purified by silica gel chromatography (35 to 60% ethyl acetatein iso-hexanes) to afford the title compound (266 mg, 85%).

To a solution of 58b (266 mg, 0.854 mmol) in tetrahydrofuran (2 mL) wasadded methanol (1 mL), water (1 mL), and lithium hydroxide hydrate (70.8mg, 1.69 mmol). The resulting mixture was stirred for 3 h and was thenquenched with 1 M aqueous hydrochloric acid (1.8 mL, 1.8 mmol).Volatiles were removed in vacuo, and the resulting solid was suspendedin toluene. Volatiles were removed in vacuo, and the resulting yellowsolid1-{(E)-2-[6-((R)-1-hydroxy-ethyl)-isoquinolin-3-yl]-vinyl}-cyclopropanecarboxylicacid was used without further purification. A solution of(S)-1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methylbutyrylamino)-propionyl]-hexahydropyridazine-3-carboxylicacid 2,2,2-trichloroethyl ester (486 mg, 0.914 mmol) in dichloromethane(9.4 mL) was cooled in an ice water bath under argon. Trimethylsilyltrifluoromethanesulfonate (370 mg, 1.7 mmol) was added dropwise, and theresulting solution was stirred for 4 h. The reaction was quenched withN,N-diisopropylethylamine (360 mg, 2.7 mmol) and methanol (2.5 mL). Themixture was concentrated in vacuo and the resulting residue wasredissolved and concentrated from toluene (2×15 mL). The resulting crudeamine was used without further purification. To a solution of crude1-{(E)-2-[6-((R)-1-hydroxy-ethyl)-isoquinolin-3-yl]-vinyl}-cyclopropanecarboxylicacid (ca. 0.854 mmol) in N,N-dimethylformamide (3.5 mL) under argon wasadded N,N-diisopropylethylamine (560 mg, 4.3 mmol) and2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (427 mg, 1.12 mmol). The resultingmixture was stirred for 2 min, at which time crude(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester from the previous step was added as asolution in N,N-dimethylformamide (2.5 mL), washing with two additionalportions of N,N-dimethylformamide (1.5 mL each). The reaction wasstirred for 45 min and was diluted with ethyl acetate (100 mL) and water(150 mL). The phases were separated, and the aqueous phase was extractedwith ethyl acetate (100 mL). The combined organic phases were washedwith water (75 mL), and the second aqueous layer was extracted withethyl acetate (75 mL). The combined organics were dried over anhydrousmagnesium sulfate, filtered, and concentrated. The crude residue waspurified by silica gel chromatography (35 to 60% acetone in iso-hexanes,continuous gradient) to afford title compound (467 mg, 78% over 2 steps)as a colorless oil.

To a solution of 58c (467 mg, 0.67 mmol) in tetrahydrofuran (6 mL) wasadded methanol (3 mL), water (3 mL), and lithium hydroxide hydrate (113mg, 2.69 mmol). The mixture was stirred for 1.75 h at ambienttemperature and was quenched with aqueous 1 M hydrochloric acid (2.8 mL,2.8 mmol). The resulting solution was concentrated in vacuo, and thecrude product was suspended and concentrated from acetonitrile (5×20 mL)to afford 530 mg of a pale yellow solid that was used without furtherpurification. Under argon, 2-methyl-6-nitrobenzoic anhydride (283 mg,0.82 mmol) and 4-dimethylaminopyridine (307 mg, 2.51 mmol) weredissolved in 1,2-dichloroethane (100 mL). The resulting solution washeated to 50° C., and a portion of the crude seco-acid (260 mg, ca. 0.33mmol) was added dropwise via syringe as a solution inN,N-dimethylformamide (3.5 mL) and 1,2-dichloroethane (10 mL) over 6 h.An additional wash of N,N-dimethylformamide (1 mL) was then added in thesame manner. After stirring an additional 1.25 h, the reaction mixturewas concentrated to −35 mL in vacuo. The solution was diluted with ethylacetate (100 mL) and washed with water (100 mL). The aqueous phase wasextracted with ethyl acetate (75 mL), and the combined organic phaseswere washed with water (50 mL). The second aqueous phase was extractedwith ethyl acetate (50 mL), and the combined organics were dried overmagnesium sulfate, filtered and concentrated. The crude residue waspurified by silica gel chromatography (35 to 60 and then to 100% acetonein iso-hexanes) to afford 101 mg of impure product containing the titlecompound. This material was purified by silica gel chromatography (0 to5% methanol in ethyl acetate) followed by reverse-phase HPLC (5 to 100%acetonitrile/water+0.1% trifluoroacetic acid) to afford the titlecompound as its trifluoroacetic acid salt (37 mg, 17% over 2 steps). ¹HNMR (400 MHz, CD₃OD) δ9.55 (s, 1H), 8.37 (d, J=8.6 Hz, 1H), 8.06 (s,1H), 7.89 (s, 1H), 7.85 (d, J=8.6 Hz, 1H), 6.69 (d, J=16.4 Hz, 1H), 6.54(d, J=16.4 Hz, 1H), 6.11 (q, J=6.7 Hz, 1H), 5.75-5.67 (m, 1H), 4.46-4.36(m, 1H), 4.36-4.28 (m, 1H), 3.90-3.78 (m, 1H), 2.80-2.68 (m, 1H),2.05-1.86 (m, 3H), 1.80-1.68 (m, 5H), 1.66-1.57 (m, 5H), 1.40-1.30 (m,1H), 1.30-1.21 (m, 1H), 1.03-0.93 (m, 4H), 0.90 (d, J=6.7 Hz, 3H). LCMS(m/z) 548.4 [M+H], Tr=2.79 min.

Example 59

Into an oven dried, argon purged flask were placed oil-free potassiumhydride (from 1740 mg of ca. 30% dispersion in mineral oil, ca. 13mmol), anhydrous tetrahydrofuran (10 mL) and hexane (1 mL). The flaskwas repurged with argon and trichloroethylene (900 μL, 1.32 g, 10 mmol)was added followed by dry methanol (10 μL, 7.9 mg, 0.25 mmol). Thismixture was stirred at RT for two h. After this time, hexane (10 mL) wasadded and the resulting solution was immediately used in the subsequentstep.

Into an oven dried, argon purged flask tetrahydrofuran (50 mL) was addedand the solution was cooled with an ice bath. A 1.8 M solution oflithium diisopropylamide (7.2 mL, 13 mmol) intetrahydrofuran/heptane/ethylbenzene was added. The resulting solutionwas cooled to −78° C., and treated dropwise with methyltetrahydro-2H-pyran-4-carboxylate (1.20 mL. 1.30 g, 9 mmol) followed byhexamethylphosphoramide (1.56 mL, 1.61 g, 9 mmol). The resultingsolution was warmed to 0° C., stirred for 20 min., cooled to −78° C.,and treated dropwise with pre-cooled (0° C.) solution of1,2-dichloro-ethyne (ca. 10 mmol). The reaction mixture was stirred at−78° C. for 30 min and then allowed to warm to RT. After 4 h at RT, thereaction mixture was poured into crushed ice and extracted with diethylether (200 mL) (5 mL of brine was added to support the separation). Theorganic phase was separated and washed with water (200 mL). This waterphase was extracted with diethyl ether (100 mL). The combined organicfractions were washed with brine (100 mL), dried over magnesium sulfate,filtered through a 2 cm layer of silica gel (silica gel layer was washedwith 50 mL of ethyl acetate), and then concentrated under reducedpressure. The crude product was subjected to silica gel chromatography(gradient from 0-15% ethyl acetate in iso-hexanes) to afford the titlecompound (1.22 g, 67%) as a colorless oil. R_(f)=0.48, 30% ethyl acetatein iso-hexanes, phosphomolybdic acid in ethanol.

59b (1.01 g, 5 mmol) and copper powder (1.6 g, 25 mmol) were suspendedin tetrahydrofuran (100 mL). Acetic acid (15 mL) was added and thereaction mixture was heated to 60° C. for 3 h. After this time, thereaction mixture was poured onto water (copper powder was filtered offwith the use of the filtration paper) and extracted with diethyl ether(3×50 mL). The combined organic extracts were washed with saturatedsolution of ammonium chloride (3×50 mL), with saturated solution ofsodium bicarbonate (2×50 mL) and with water (50 mL). This water phasewas extracted with diethyl ether (50 mL). Combined organic extracts werewashed with brine (50 mL) and dried over magnesium sulfate, filteredthrough a 2 cm layer of silica gel (silica gel layer was washed with 50mL of ethyl acetate), and concentrated under reduced pressure. Afterdrying under high vacuum for one day, the title compound was isolated(0.84 g, quantitative yield) as a colorless oil. R_(f)=0.37, 30% ethylacetate in iso-hexanes, phosphomolybdic acid in ethanol.

Into an oven dried, argon purged flask were placed 59c (0.84 g, 5 mmol)and dichloromethane (2 mL). This mixture was cooled to 0° C.Pinacolborane (0.96 g, 7.5 mmol) was then added dropwise via syringe.After the mixture was stirred for 1 min., it was transferred by asyringe into another oven dried, argon purged flask, immersed in an icebath and protected from light, containing zirconocene dichloride (0.13g, 0.5 mmol). An additional portion of dichloromethane (2 mL) was usedto complete the quantitative transfer. After this the mixture was warmedto ambient temperature, it was stirred in the dark for 72 h to achievefull conversion. The reaction mixture was diluted with ethyl acetate (50mL) and carefully quenched with water (1 ml). Water (50 mL) was addedand the organic and aqueous phases were separated. The aqueous phase wasextracted with ethyl acetate (3×40 mL). The combined organic phases weredried over magnesium sulfate, filtered and evaporated to afford thecrude residue which was dissolved in iso-hexanes (50 mL) and extractedwith 5-10% aqueous methanol (3×50 mL) and with brine (50 mL). The hexanephase was dried over magnesium sulfate. The title compound was isolatedas the white crystalline compound after evaporation (1.42 g, 93%).R_(f)=0.38, 30% ethyl acetate in iso-hexanes, iodine vapor.

Into an oven dried, argon purged flask were placed(R)-1-(3-chloro-isoquinolin-6-yl)-ethanol (0.83 g, 4 mmol), 59d (1.40 g,4.7 mmol), PdCl₂(PCy₂(p-NMe₂Ph))₂(bis[(dicyclohexyl)(4-dimethylaminophenyl)phosphine] palladium(II)chloride) (173 mg, 0.21 mmol) and potassium phosphate tribasic (2.64 g,12.4 mmol). The flask was sealed with a septum cap and was re-purgedwith argon. N,N-Dimethylformamide (10 mL) was added and, and theresulting reaction mixture was vigorously stirred in an oil bathpre-heated to 80° C. After 2 h, the reaction was cooled to ambienttemperature and was diluted with ethyl acetate (100 mL) and water (100mL). The phases were separated, and the aqueous phase was extracted withethyl acetate (2×50 mL). The combined organic phases were dried overmagnesium sulfate, filtered, and concentrated to afford a crude residuethat was purified by silica gel chromatography (gradient from 0-40%ethyl acetate and methanol (4/1) in iso-hexanes) to afford the titlecompound (1.06 g, 78%) as a colorless oil after evaporation. R_(f)=0.48,iso-hexanes/ethyl acetate/methanol (6/4/1).

To a solution of 59e (1.02 g, 3 mmol) in tetrahydrofuran (8 mL) wasadded methanol (4 mL), water (4 mL) and lithium hydroxide hydrate (0.15g, 6.3 mmol). The resulting mixture was stirred at RT for 10 h andquenched with 1 M hydrochloric acid (6.5 mL, 6.5 mmol). The resultingsolution was concentrated to a crude residue which was co-evaporatedtwice with tetrahydrofuran (20 mL), twice with anhydrous acetonitrile(20 mL) and twice with anhydrous toluene (20 mL). The resulting whitesolid was dried under high vacuum overnight and used without furtherpurification (1.24 g, quantitative yield).

A solution of 1e (0.53 g, 1 mmol) in dichloromethane (10 mL) was cooledin an ice water bath. Trimethylsilyl trifluoromethanesulfonate (0.69 g,1.80 mmol) was added dropwise at 0° C. under argon, and the resultingsolution was stirred at RT for 30 min. The reaction mixture wasevaporated to dryness and the resulting crude residue was dissolved inanhydrous acetonitrile (12 mL) under argon. The reaction mixture wasstirred at 0° C., 59f (371 mg, 0.9 mmol) and N,N-diisopropylethylamine(517 mg, 4 mmol) were added followed by2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (570 mg, 1.5 mmol). The reactionmixture was stirred at RT for 48 h. The solvent was evaporated, theresidue was dissolved in ethyl acetate (50 mL) and the solution waswashed with 20% water solution of citric acid (2×50 mL), water (50 mL)and brine (50 mL), dried over magnesium sulfate, filtered andevaporated. The residue was purified by silica gel chromatography(gradient from 0-40% ethyl acetate and methanol (4/1) in iso-hexanes) toafford the title compound (560 mg, 84%) as a white solid afterevaporation. R_(f)=0.13, iso-hexanes/ethyl acetate/methanol (6/4/1).

To a solution of 59g (371 mg, 0.5 mmol) in tetrahydrofuran (8 mL) wasadded methanol (4 mL), water (4 mL) and lithium hydroxide hydrate (36mg, 1.5 mmol). The mixture was stirred for 2 h at ambient temperatureand was quenched with aqueous 1 M hydrochloric acid (1.6 mL, 1.6 mmol).The resulting solution was concentrated to a crude residue which wasco-evaporated twice with tetrahydrofuran (20 mL), twice with anhydrousacetonitrile (20 mL) and twice with anhydrous toluene (20 mL). Theresulting white solid was dried under high vacuum overnight and it wasused without further purification (365 mg, quantitative yield). Intooven dried, argon purged flask were placed 2-methyl-6-nitrobenzoicanhydride (258 mg, 0.75 mmol), 4-dimethylaminopyridine (275 mg, 2.25mmol) and anhydrous 1,2-dichloroethane (150 mL). The resulting solutionwas heated at 50° C., and the crude seco-acid was added dropwise viasyringe as a solution in dry N,N-dimethylformamide (10 mL) over 12 h. Anadditional portion of dry N,N-dimethylformamide (2×5 mL) was used tocomplete the quantitative transfer. After stirring for additional 2 h at50° C., the reaction mixture was transferred to a separatory funnel andwashed with water (100 mL, 5 mL of brine was added to support theseparation). The aqueous phase was extracted with dichloromethane (50mL). Combined organic extracts were washed with brine (50 mL) and driedover magnesium sulfate, filtered and concentrated under reducedpressure. The resulting residue was dissolved in ethyl acetate (100 mL)and was washed with water (100 mL, 5 mL of brine was added to supportthe separation). The aqueous phase was extracted with ethyl acetate (50mL). Combined organic extracts were washed with water (100 mL, 5 mL ofbrine was added to support the separation). Resulting aqueous phase wasextracted with ethyl acetate (50 mL). Combined organic extracts werewashed with brine (50 mL) and dried over magnesium sulfate, filtered andconcentrated under reduced pressure. The residue was purified by silicagel chromatography (gradient from 0-40% ethyl acetate and methanol (4/1)in iso-hexanes) to afford the title compound (72 mg, 24%) as a whitesolid after evaporation. R_(f)=0.42, 10% methanol in dichloromethane. ¹HNMR (400 MHz, CD₃OD): δ9.04 (s, 1H), 7.95 (d, J=8.5 Hz, 1H), 7.76 (s,1H), 7.47 (dd, J=8.5, 1.5 Hz, 1H), 7.31 (s, 1H), 6.43 (m, 2H), 5.93 (q,J=6.6 Hz, 1H), 5.44-5.36 (m, 1H), 4.33-4.23 (m, 2H), 3.84 (dt, J=11.6,4.1 Hz, 1H), 3.76-3.69 (m, 1H), 3.68-3.61 (m, 2H), 3.48 (m, 1H),2.65-2.56 (m, 1H), 2.16-2.07 (m, 2H), 2.03-1.93 (m, 1H), 1.91-1.82 (m,2H), 1.79e (m, 1H), 1.67-1.62 (m, 3H), 1.59 (d, J=6.6 Hz, 3H), 1.51 (d,J=7.2 Hz, 3H), 0.90 (d, J=6.7 Hz, 3H), 0.85 (d, J=6.7 Hz, 3H). LCMS(m/z) 592.3 [M+H]′ Tr=3.12 min.

Example 60

A solution of Compound 59 (10 mg, 0.017 mmol) in a mixture of ethylacetate (4 mL) and tetrahydrofuran (4 mL) containing 10% palladium oncarbon (10 mg) was hydrogenated at RT and at atmospheric pressure ofhydrogen for 3 h. The reaction mixture was filtered through Celite andthe filter pad was washed with tetrahydrofuran (10 mL). The filtrate wasevaporated to afford the title compound (10 mg, quantitative yield) as awhite solid. R_(f)=0.47, 10% methanol in dichloromethane. ¹H NMR (400MHz, CD₃OD): δ9.02 (s, 1H), 7.94 (d, J=8.5 Hz, 1H), 7.81 (s, 1H), 7.44(d, J=8.5 Hz, 1H), 7.20 (s, 1H), 6.02 (q, J=6.5 Hz, 1H), 5.78-5.71 (m,1H), 5.24 (m, 1H), 4.33-4.23 (m, 2H), 3.83-3.61 (m, 6H), 3.52 (m, 1H),2.71-2.66 (m, 1H), 2.43-2.36 (m, 2H), 2.14-2.05 (m, 2H), 2.08-1.91 (m,1H), 1.90-1.82 (m, 2H), 1.76 (m, 1H), 1.67-1.62 (m, 2H), 1.57 (d, J=6.6Hz, 3H), 1.54 (d, J=7.2 Hz, 3H), 0.96 (d, J=6.7 Hz, 3H), 0.88 (d, J=6.7Hz, 3H). LCMS (m/z) 594.3 [M+H]′ Tr=2.72 min.

Example 61

A solution of 1e (1064 mg, 2 mmol) in dichloromethane (30 mL) was cooledin an ice water bath. Trimethylsilyl trifluoromethanesulfonate (666 mg,3 mmol) was added dropwise at 0° C. under argon, and the resultingsolution was stirred at RT for 30 min. The reaction mixture wasevaporated to dryness and the resulting crude residue was dissolved inanhydrous acetonitrile (25 mL) under argon. The reaction mixture wasstirred at 0° C., hex-5-enoic acid (251 mg, 2.2 mmol) andN,N-diisopropylethylamine (1034 mg, 8 mmol) were added followed by2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (1065 mg, 2.8 mmol). The reactionmixture was stirred at RT for 48 h. The solvent was evaporated, theresidue was dissolved in ethyl acetate (200 mL) and the solution waswashed with 20% water solution of citric acid (2×150 mL), water (150 mL)and brine (150 mL), dried over magnesium sulfate, filtered andevaporated. The residue was purified by silica gel chromatography(gradient from 0-40% ethyl acetate and methanol (4/1) in iso-hexanes) toafford the title compound (864 mg, 82%) as a white solid afterevaporation. R_(f)=0.35, iso-hexanes/ethyl acetate/methanol (6/4/1).

To a solution of 61a (830 mg, 1.57 mmol) in tetrahydrofuran (40 mL) wereadded water (10 mL) and lithium hydroxide hydrate (57 mg, 2.38 mmol).The mixture was stirred for 2 h at ambient temperature and then filteredthrough a 5 cm layer of DOWEX D50×8 resin in H⁺ cycle (resin firstlywashed with water). Resin was washed with additional water (50 mL).Filtrates were collected, concentrated under reduced pressure andco-evaporated twice with toluene (10 mL). After drying under high vacuumfor one day, the title compound was isolated (590 mg, 95%) as a whitesolid. R_(f)=0.4, 30% methanol in dichloromethane.

Into an oven dried, argon purged flask were added 61b (238 mg, 0.60mmol) and (R)-1-(3-vinyl-isoquinolin-6-yl)-ethanol (120 mg, 0.6 mmol).The flask was sealed and the reaction mixture was repurged twice withargon. Anhydrous dichloromethane (10 mL) was added and the reactionmixture was repurged twice with argon.N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (148 mg,0.77 mmol) was added followed by 4-dimethylaminopyridine (67 mg, 0.55mmol). Reaction mixture was quickly repurged twice with argon and wasstirred at RT for 12 h. The reaction mixture was diluted withdichloromethane (100 mL) and the solution was washed with 20% watersolution of citric acid (2×150 mL), water (150 mL) and brine (150 mL),dried over magnesium sulfate, filtered and evaporated. The residue waspurified by silica gel chromatography (gradient from 0-40% ethyl acetateand methanol (4/1) in iso-hexanes) to afford the title compound (307 mg,89%) as a white solid after evaporation. R_(f)=0.29, iso-hexanes/ethylacetate/methanol (6/4/1).

A solution of 61c (209 mg, 0.362 mmol) in toluene (150 mL) was stirredat RT under argon. Hoveyda-Grubbs 2^(nd) generation catalyst (23 mg,0.036 mmol) was added and the reaction mixture was heated at refluxunder argon for 30 min. Reaction mixture was cooled to RT and ethylacetate (50 mL) was added. This solution was washed twice with aqueoussolution of tris(hydroxymethyl)phosphine (372 mg, 3 mmol in 100 mL ofwater), with water (2×50 mL) and with brine (50 mL). The organic phasewas dried over magnesium sulfate, filtered and evaporated. The residuewas purified by silica gel chromatography (gradient from 0-40% ethylacetate and methanol (4/1) in iso-hexanes) to afford the title compound(125 mg, 63%) as a white solid after evaporation. R_(f)=0.25,iso-hexanes/ethyl acetate/methanol (6/4/1). ¹H NMR (400 MHz, CD₃OD):δ9.00 (s, 1H), 7.91 (d, J=8.4 Hz, 1H), 7.77 (s, 1H), 7.41 (d, J=8.4 Hz,1H), 7.23 (s, 1H), 6.64 (m, 1H), 6.29 (d, J=15.4 Hz, 1H), 5.97 (q, J=5.9Hz, 1H), 5.53 (q, J=7.0 Hz, 1H), 4.37-4.18 (m, 2H), 3.65 (d, J=9.8 Hz,1H), 2.65 (t, J=12.3 Hz, 1H), 2.36-2.18 (m, 4H), 1.98 (m, 1H), 1.94-1.73(m, 3H), 1.71-1.57 (m, 3H), 1.55 (d, J=5.6 Hz, 6H), 0.83 (d, J=6.6 Hz,3H), 0.78 (d, J=6.6 Hz, 3H). LCMS (m/z) 550.2 [M+H]′ Tr=2.55 min.

Example 62

A solution of Compound 61 (10 mg, 0.018 mmol) in a mixture of ethylacetate (4 mL) and tetrahydrofuran (4 mL) containing 10% palladium oncarbon (10 mg) was hydrogenated at RT and at atmospheric pressure ofhydrogen for 3 h. The reaction mixture was filtered through Celite andthe filter pad was washed with tetrahydrofuran (10 mL). The filtrate wasevaporated to afford the title compound (9 mg, 89%) as a white solid.R_(f)=0.16, iso-hexanes/ethyl acetate/methanol (6/4/1). ¹H NMR (400 MHz,CD₃OD) δ9.02 (s, 1H), 7.93 (d, J=8.7 Hz, 1H), 7.62 (s, 1H), 7.47 (s,1H), 7.44 (d, J=8.7 Hz, 1H), 6.02-5.93 (m, 1H), 5.39 (m, 1H), 4.03 (m,1H), 3.69 (m, 1H), 2.84 (m, 2H), 2.27 (s, 1H), 2.10 (m, 1H), 1.97 (m,1H), 1.85 (m, 2H), 1.76 (m, 2H), 1.62 (m, 4H), 1.54 (d, J=5.1 Hz, 3H),1.39 (d, J=6.6 Hz, 3H), 1.19 (m, 4H), 0.78 (d, J=5.0 Hz, 3H), 0.73 (d,J=5.8 Hz, 3H). LCMS (m/z) 552.3 [M+H]′ Tr=2.10 min.

Example 63

A solution of 1e (1064 mg, 2 mmol) in dichloromethane (30 mL) was cooledin an ice water bath. Trimethylsilyl trifluoromethanesulfonate (666 mg,3 mmol) was added dropwise at 0° C. under argon, and the resultingsolution was stirred at RT for 30 min. The reaction mixture wasevaporated to dryness and the resulting crude residue was dissolved inanhydrous acetonitrile (25 mL) under argon. The reaction mixture wasstirred at 0° C., hept-6-enoic acid (281 mg, 2.2 mmol) andN,N-diisopropylethylamine (1034 mg, 8 mmol) were added followed by2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (1065 mg, 2.8 mmol). The reactionmixture was stirred at RT for 48 h. The solvent was evaporated, theresidue was dissolved in ethyl acetate (200 mL) and the solution waswashed twice with 20% water solution of citric acid (2×150 mL), water(150 mL) and brine (150 mL), dried over magnesium sulfate, filtered andevaporated. The residue was purified by silica gel chromatography(gradient from 0-40% ethyl acetate+methanol (4/1) in iso-hexanes) toafford the title compound (817 mg, 75%) as a white solid afterevaporation. R_(f)=0.37, iso-hexanes/ethyl acetate/methanol (6/4/1).

To a solution of 63a (790 mg, 1.46 mmol) in tetrahydrofuran (40 mL) wasadded water (10 mL) and lithium hydroxide hydrate (52 mg, 2.19 mmol).The mixture was stirred for 2 h at ambient temperature and then filteredthrough a 5 cm layer of DOWEX D50×8 resin in H⁺ cycle (resin firstlywashed with water). Resin was washed with additional water (50 mL). Thefiltrates were collected, concentrated under reduced pressure andco-evaporated twice with toluene (10 mL). After drying under high vacuumfor one day, the title compound was isolated (583 mg, 97%) as a whitesolid. R_(f)=0.4, 30% methanol in dichloromethane.

Into an oven dried, argon purged flask were added 63b (276 mg, 0.67mmol) and (R)-1-(3-vinyl-isoquinolin-6-yl)-ethanol (134 mg, 0.67 mmol).The flask was sealed and the reaction mixture was repurged twice withargon. Anhydrous dichloromethane (10 mL) was added and the reactionmixture was repurged twice with argon.N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (168 mg,0.87 mmol) was added followed by 4-dimethylaminopyridine (75 mg, 0.62mmol). The reaction mixture was quickly repurged twice with argon andwas stirred at RT for 12 h. The reaction mixture was diluted withdichloromethane (100 mL) and the solution was washed with 20% watersolution of citric acid (2×150 mL), water (150 mL) and brine (150 mL),dried over magnesium sulfate, filtered and evaporated. The residue waspurified by silica gel chromatography (gradient from 0-40% ethyl acetateand methanol (4/1) in iso-hexanes) to afford the title compound (316 mg,79%) as a white solid after evaporation. R_(f)=0.30, iso-hexanes/ethylacetate/methanol (6/4/1).

A solution of 63c (255 mg, 0.431 mmol) in toluene (200 mL) was stirredat RT under argon. Hoveyda-Grubbs 2^(nd) generation catalyst (27 mg,0.043 mmol) was added and the reaction mixture was heated at refluxunder argon for 30 min. Reaction mixture was cooled to RT and ethylacetate (50 mL) was added. This solution was washed twice with aqueoussolution of tris(hydroxymethyl)phosphine (372 mg, 3 mmol in 100 mL ofwater), twice with water (50 mL) and with brine (50 mL). The organicphase was dried over magnesium sulfate, filtered and evaporated. Theresidue was purified by by silica gel chromatography (gradient from0-40% ethyl acetate and methanol (4/1) in iso-hexanes) to afford thetitle compound (76 mg, 31%) as a white solid after evaporation.R_(f)=0.20, iso-hexanes/ethyl acetate/methanol (6/4/1). ¹H NMR (400 MHz,CD₃OD): δ9.01 (s, 1H), 7.93 (d, J=8.5 Hz, 1H), 7.69 (s, 1H), 7.57 (s,1H), 7.43 (d, J=8.5 Hz, 1H), 6.55 (m, 1H), 5.95 (m, 1H), 5.43 (m, 1H),5.23 (m, 3H), 4.32 (m, 1H), 4.20 (m, 1H), 3.67 (m, 1H), 2.69 (m, 1H),2.30 (m, 2H), 2.13 (m, 2H), 1.96 (m, 3H), 1.83 (m, 1H), 1.64 (m, 3H),1.56 (d, J=6.7 Hz, 3H), 1.44 (d, J=7.4 Hz, 3H), 0.92-0.72 (m, 6H). LCMS(m/z) 564.4 [M+H], Tr=2.60 min.

Example 64

A solution of Compound 63 (15 mg, 0.027 mmol) in a mixture of ethylacetate (4 mL) and tetrahydrofuran (4 mL) containing 10% palladium oncarbon (10 mg) was hydrogenated at RT and at atmospheric pressure ofhydrogen for 3 h. The reaction mixture was filtered through Celite andthe filter pad was washed with tetrahydrofuran (10 mL). The filtrate wasevaporated to afford the title compound (13 mg, 87%) as a white solid.R_(f)=0.11, iso-hexanes/ethyl acetate/methanol (6/4/1). ¹H NMR (400 MHz,CD₃OD) δ9.02 (s, 1H), 7.95 (d, J=8.7 Hz, 1H), 7.68 (s, 1H), 7.50 (s,1H), 7.45 (d, J=8.7 Hz, 1H), 5.93 (q, J=6.3 Hz, 1H), 5.20 (m, 1H), 4.21(m, 1H), 4.07 (d, J=8.0 Hz, 1H), 3.66 (m, 1H), 2.83 (m, 4H), 2.25 (m,1H), 2.07 (m, 1H), 1.94 (m, 2H), 1.80e (m, 1H), 1.63 (m, 3H), 1.55 (d,J=6.6 Hz, 3H), 1.51-1.42 (m, 2H), 1.37 (d, J=7.0 Hz, 3H), 1.25 (m, 4H),0.83 (d, J=6.7 Hz, 6H). LCMS (m/z) 566.3 [M+H], Tr=2.27 min.

Example 65

To a solution of (R)-1-(3-chloro-isoquinolin-6-yl)-ethanol (250 mg, 1.21mmol), and potassium phosphate tribasic (770 mg, 3.63 mmol) incyclopentyl methy ether (4.5 mL) and water (1.5 mL) preheated to 90° C.under an argon atmosphere were added3-(2-methoxy-2-oxoethyl)phenylboronic acid, pinacol ester (Combi-Blocks,387 mg, 1.33 mmol) and (A-^(ca)Phos)₂PdCl₂ (49 mg, 60 μmol). After 17 h,the reaction was allowed to cool to 23° C., and was partitioned betweendichloromethane (50 mL) and saturated aqueous sodium bicarbonatesolution (50 mL). The phases were split and the aqueous layer wasextracted with dichloromethane (50 mL). The combined organic layers weredried over anhydrous sodium sulfate, and were concentrated under reducedpressure. The crude residue was purified by silica gel chromatography(24 g Combiflash HP Gold Column, 0-100% ethyl acetate/iso-hexanesgradient) to afford the title compound (321 mg, 83%) as a faint yellowoil.

To a solution of 65a (320 mg, 1.00 mmol) in tetrahydrofuran (3 mL) andwater (2 mL) was added lithium hydroxide (26 mg, 1.1 mmol) at 23° C.under an argon atmosphere. After 3 h, the reaction mixture wasconcentrated under reduced pressure and the crude residue was purifiedby silica gel chromatography (24 g Combiflash HP Gold Column, 0-20%methanol/dichloromethane gradient) to afford the title compound (64.2mg, 21%) as a colorless oil. R_(f)=0.5 (20% methanol in dichloromethane)I₂/silica stain.

To a solution of 1e (104 mg, 0.21 mmol) in dichloromethane (1.05 mL) wasadded trimethylsilyl trifluoromethanesulfonate (70 mg, 1.07 mmol) at 0°C. under an argon atmosphere. After 1 h, the reaction mixture wasconcentrated under reduced pressure. The resulting residue was dilutedwith acetonitrile (1.05 mL) and2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (79.8 mg, 0.210 mmol),N,N-diisopropylethylamine (140 μL, 0.840 mmol), and 65b (64 mg, 0.21mmol) were sequentially added at 23° C. under an argon atmosphere. After18 h, the reaction mixture was concentrated under reduced pressure, andthe crude residue was purified by silica gel chromatography to affordthe title compound (140 mg, 93%) as a faint yellow oil.

To a solution of 65c (140 mg, 0.195 mmol) in tetrahydrofuran (0.9 mL)and water (0.3 mL) was added lithium hydroxide hydrate (4.6 mg, 0.195mmol) at 23° C. under an argon atmosphere. After 3 h, the reactionmixture was concentrated under reduced pressure to afford the titlecompound (131 mg, 99%) as a white solid lithium carboxylate salt.

To a solution of benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate (35.3 mg, 68 μmol) and 4-dimethylaminopyridine (62.3mg, 510 μmol) in dichloromethane (5.7 mL) was added 65d (10 mg, 17 μmol)at 23° C. under an argon atmosphere. After 16 h, the reaction mixturewas concentrated under reduced pressure and the crude residue waspurified by preparative HPLC (Gemini 5u C18 110 Å column, 5-100%acetonitrile/water, 0.1% trifluoroacetic acid modifier) to afford thetitle compound (6.0 mg, 52%) as a white solid trifluoroacetic acid salt.¹H NMR (400 MHz, CD₃OD) δ9.70 (s, 1H), 8.58 (s, 1H), 8.48 (d, J=8.6 Hz,1H), 8.32 (s, 1H), 7.97-7.87 (m, 2H), 7.79 (d, J=7.9 Hz, 1H), 7.62 (appt, J=7.7 Hz, 1H), 7.53 (d, J=7.8 Hz, 1H), 6.21 (q, J=6.6 Hz, 1H),5.84-5.71 (m, 1H), 4.45-4.29 (m, 2H), 3.96 (d, J=15.5 Hz, 1H), 3.75 (dd,J=11.1, 2.7 Hz, 1H), 3.64 (d, J=15.5 Hz, 1H), 2.74 (td, J=12.8, 3.1 Hz,1H), 2.13-1.96 (m, 2H), 1.91 (brd, J=13.0 Hz, 1H), 1.81-1.65 (m, 2H),1.67 (d, J=6.6 Hz, 3H), 1.62 (d, J=7.1 Hz, 3H), 1.01 (d, J=6.8 Hz, 3H),0.94 (d, J=6.8 Hz, 3H). HPLC Tr=3.040 min. LCMS (m/z) 572.3 [M+H],Tr=2.07 min.

Example 66

To a solution of 6-chloropyridin-3-amine (5.00 g, 38.8 mmol) in dioxane(194 mL) was added di-tert-butyl dicarbonate (10.2 g, 46.7 mmol) at 23°C. under an argon atmosphere, and the resulting mixture was heated to100° C. After 17 h, the reaction mixture was allowed to cool to 23° C.,and was diluted with water (500 mL). The resulting mixture was extractedwith ethyl acetate (2×500 mL), and the combined organic extracts weredried over anhydrous sodium sulfate and were concentrated under reducedpressure. The crude residue was purified by silica gel chromatography(120 g Combiflash HP Gold Column, 0-100% ethyl acetate/iso-hexanesgradient) to afford the title compound (7.69 g, 87%) as a colorless oil.

To a solution of 66a (2.00 g, 8.80 mmol) and tetramethylethylenediamine(2.70 mL, 18.0 mmol) in diethyl ether (44 mL) was added n-butyllithium(2.5 M in hexanes, 7.2 mL, 18.0 mmol) at −78° C. under an argonatmosphere. After 10 min, the resulting mixture was allowed to warm to−15° C. over a 50 min period. The reaction mixture was cooled to −78°C., and N,N-dimethylformamide (1.9 g, 26 mmol) was added via syringe.After 30 min, the reaction mixture was quenched with saturated aqueousammonium chloride solution (20 mL) and was allowed to warm to 23° C. Theresulting mixture was extracted with ethyl acetate (2×20 mL), and thecombined organic layers were dried over anhydrous sodium sulfate andwere concentrated under reduced pressure. The crude residue was purifiedby silica gel chromatography (120 g Combiflash HP Gold Column, 0-100%ethyl acetate/iso-hexanes gradient) to afford the title compound (849mg, 38%) as colorless oil.

To a solution of 66b (258 mg, 1.00 mmol) and(E)-4-(dimethylamino)but-3-en-2-one (452 mg, 4.00 mmol) in 1,4-dioxane(10 mL) was added p-toluenesulfonic acid monohydrate (761 mg, 4 mmol) at23° C. under an argon atmosphere and the resulting mixture was heated to80° C. After 2 h, the reaction mixture was allowed to cool to 23° C. andwas partitioned between saturated aqueous sodium bicarbonate solution(200 mL) and ethyl acetate (200 mL). The layers were split and theaqueous layer was extracted with ethyl acetate (200 mL). The combinedorganic layers were dried over anhydrous sodium sulfate and wereconcentrated under reduced pressure. The crude residue was purified bysilica gel chromatography to afford the title compound (107 mg, 51%) asan off-white solid.

Dichloro (p-cymene) ruthenium(II) dimer (5 mg, 8 μmol) and(1R,2R)-(−)-N-p-tosyl-1,2-diphenylethylenediamine (7 mg, 19 μmol) weresuspended in degassed water (6 mL) and the mixture was degassed withargon for 15 min. The mixture was stirred at 70° C. under argon for 90min. The resulting yellow solution was cooled to RT. 66c (329 mg, 1.56mmol), sodium formate (543 mg, 7.98 mmol) and degassed tetrahydrofuran(1 mL) were added and the reaction mixture was degassed for 10 min. Thereaction mixture was vigorously stirred at 40° C. for 2.5 h. Thereaction mixture was cooled to RT and was extracted with ethyl acetate(20 mL). The organic layer was separated, washed with water (20 mL),brine (20 mL), dried over anhydrous magnesium sulfate, and concentratedunder reduced pressure. The residue was purified by silica gelchromatography to afford the title compound (180 mg, 54%) as a solid.

To a suspension of potassium phosphate tribasic (550 mg, 2.59 mmol) incyclopentyl methyl ether (4.5 mL) and water (3 mL) was added 66d (180mg, 0.89 mmol) and heated to 90° C. At this temperature,(A-^(ca)Phos)₂PdCl₂(35 mg, 43 μmol) was added and stirred for 2 min. Asolution of(E)-2,2-dimethyl-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-but-3-enoicacid methyl ester (286 mg, 1.12 mmol) in cyclopentyl methyl ether (4.5mL) was added dropwise and stirred for 19 h at 90° C. The reactionmixture was cooled to RT, diluted with ethyl acetate (40 mL) and water(20 mL). The layers were separated and the aqueous layer was extractedwith ethyl acetate (2×20 mL). The combined organic layers were driedover anhydrous magnesium sulfate, concentrated and the resulting cruderesidue was purified via silica gel chromatography (24 g SiO₂ Isco RfGold Column, 0-100% ethyl acetate/iso-hexanes gradient) to afford thetitle compound (95 mg, 37%) as a pale brown solid.

To a solution of 66e (95 mg, 0.32 mmol) in tetrahydrofuran (1.8 mL),methanol (0.6 mL) and water (0.6 mL) was added lithium hydroxide hydrate(15 mg, 0.63 mmol) at 23° C. After 16 h, the resulting mixture wasconcentrated under reduced pressure and residual solvents were removedazeotropically by addition of toluene (5 mL) followed by concentrationunder reduced pressure (2×) to afford the title compound. This was usedin the subsequent amide coupling without further purification.

To a solution of 1e (212 mg, 0.40 mmol) in dichloromethane (3 mL) wasslowly added trimethylsilyl trifluoromethanesulfonate (106 μL, 0.60mmol) at 0° C. under an argon atmosphere. After 1 h, the resultingmixture was concentrated under reduced pressure and was used in thesubsequent amide coupling without further purification. To a solution of66f (90.5 mg, 0.32 mmol) in acetonitrile (3 mL),2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (180 mg, 0.47 mmol),N,N-diisopropylethylamine (330 μL, 1.90 mmol) and(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (232 mg, 0.4 mmol) in acetonitrile (2mL) were added sequentially at 23° C. After 16 h, the resulting mixturewas concentrated under reduced pressure and the residue was purifieddirectly by silica gel chromatography to afford the title compound (201mg, 91%) as a solid.

To a solution of 66g (201 mg, 0.29 mmol) in tetrahydrofuran (2 mL),methanol (0.4 mL) and water (0.4 mL) was added lithium hydroxide hydrate(14 mg, 0.57 mmol) at 23° C. After 40 min, lithium hydroxide hydrate (14mg, 0.57 mmol) was added at 23° C. After 1 h, the resulting mixture wasconcentrated under reduced pressure and residual solvents were removedazeotropically by addition of toluene (2 mL) followed by concentrationunder reduced pressure (3×) to afford the title compound. This was usedin the subsequent macrolactonization without further purification.

To 2-methyl-6-nitrobenzoic anhydride (43 mg, 0.12 mmol) and4-dimethylaminopyridine (48 mh, 0.39 mmol) was added 1,2-dichloroethane(20 mL) under nitrogen atmosphere and heated to 50° C. At thistemperature, 66h (28 mg, 0.049 mmol) in N,N-dimethylformamide (1 mL) wasadded dropwise via syringe pump over 6 h. An additional wash ofN,N-dimethylformamide (0.5 mL) was then added in the same manner over 15min. After stirring an additional 1.25 h, reaction mixture was cool toRT. It was diluted with ethyl acetate (20 mL) and washed with water(2×10 mL). The organic layer was dried over anhydrous magnesium sulfateand filtered. The solvent was removed under reduced pressure and theresidue was purified by preparative HPLC (Gemini 5u C18 110 Å column,5-100% acetonitrile/water, 0.1% trifluoroacetic acid modifier) to affordthe title compound as a white powder trifluoroacetic acid salt. It waswashed with saturated solution of sodium bicarbonate to remove acidimpurities formed from 2-methyl-6-nitrobenzoic anhydride to afford thetitle compound (1.5 mg, 5%) as a white solid. ¹H NMR (400 MHz, CD₃OD)δ9.19 (s, 1H), 8.86 (d, J=2.1 Hz, 1H), 8.67 (d, J=7.5 Hz, 1H), 8.21 (s,1H), 7.59 (s, 1H), 6.89 (d, J=9.5 Hz, 1H), 6.58 (d, J=16.1 Hz, 1H), 6.46(d, J=16.1 Hz, 1H), 6.04 (q, J=6.4 Hz, 1H), 5.52-5.41 (m, 1H), 4.26 (d,J=14.4 Hz, 1H), 4.18 (app t, J=9.1 Hz, 1H), 3.65 (dd, J=11.4, 2.8 Hz,1H), 2.58 (td, J=12.9, 3.2 Hz, 1H), 1.91-1.75 (m, 3H), 1.68-1.53 (m,2H), 1.63 (d, J=6.7 Hz, 3H), 1.51 (d, J=7.3 Hz, 3H), 1.43 (s, 3H), 1.26(s, 3H), 0.87 (d, J=6.7 Hz, 3H), 0.82 (d, J=6.7 Hz, 3H).LCMS (m/z) 551.2[M+H], Tr=2.19 min.

Example 67

Methyl 2-(3-bromophenyl)-2-methylpropanoate (Pharmabridge, Doylestown,Pa., USA (190 mg, 0.74 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (206 mg,0.81 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane (27 mg, 0.04 mmol)and potassium acetate (217 mg, 2.22 mmol) were placed in a screw capvial and flushed with a vacuum and argon cycle three times. Anhydrous1,4-dioxane (4 mL) was added under argon and the resulting mixture washeated to 80° C. for 19 h. The reaction mixture was cool to RT and wasdiluted with ethyl acetate (20 mL). Celite (˜1 g) was added and filteredthrough a pad of celite. Solvents were removed under reduced pressure toafford the title compound which was used directly in the subsequentreaction.

To a suspension of potassium phosphate tribasic (445 mg, 2.10 mmol) incyclopentyl methyl ether (2.5 mL) and water (1.5 mL) was added(R)-1-(3-chloro-isoquinolin-6-yl)-ethanol (180 mg, 0.89 mmol) and thereaction mixture was heated to 90° C. At this temperature,(A-^(ca)Phos)₂PdCl₂ (28 mg, 35 μmol) was added and the reaction mixturewas stirred for 2 min. A solution of 67a (225 mg, 0.74 mmol) incyclopentyl methyl ether (2.5 mL) was added dropwise and stirred for 19h at 90° C. The reaction mixture was cool to RT, diluted with ethylacetate (30 mL) and water (15 mL). The layers were separated and theaqueous layer was extracted with ethyl acetate (2×15 mL). The combinedorganic layers were dried over anhydrous magnesium sulfate, concentratedand the resulting crude residue was purified by silica gel columnchromatography to afford the title compound (267 mg, quantitative) as apale brown solid.

To a solution of 67b (244 mg, 0.7 mmol) in tetrahydrofuran (10 mL), andwater (2.5 mL) was added lithium hydroxide hydrate (18.5 mg, 0.77 mmol)at 23° C. After 48 h, lithium hydroxide hydrate (17 mg, 0.70 mmol) wasadded to the reaction mixture. After 24 h, the reaction mixture wasconcentrated under reduced pressure and residual solvents were removedazeotropically by addition of toluene (5 mL) followed by concentrationunder reduced pressure (2×) to afford the title compound. This was usedin the subsequent amide coupling without further purification.

To a solution of 1e (397 mg, 0.75 mmol) in dichloromethane (5 mL) wasslowly added trimethylsilyl trifluoromethanesulfonate (199 μL, 1.12mmol) at 0° C. under an argon atmosphere. After 1 h, the resultingmixture was concentrated under reduced pressure to give(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester as a triflate salt. This was used inthe subsequent amide coupling without further purification. To asolution of 67c (234 mg, 0.7 mmol) in acetonitrile (7 mL),2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (532 mg, 1.4 mmol),N,N-diisopropylethylamine (730 μL, 4.2 mmol) and(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (435 mg, 0.75 mmol) in acetonitrile (3mL) were added sequentially at 23° C. After 16 h, the resulting mixturewas concentrated under reduced pressure and the residue was purifieddirectly by silica gel chromatography (40 g SiO₂ Isco Rf Gold Column,0-100% ethyl acetate/iso-hexanes gradient) to afford the title compound(230 mg, 44%) as a solid.

To a solution of 67d (94 mg, 0.13 mmol) in tetrahydrofuran (3 mL), andwater (1 mL) was added lithium hydroxide hydrate (3.1 mg, 0.13 mmol) at23° C. After 48 h, lithium hydroxide hydrate (17 mg, 0.70 mmol) wasadded to the reaction mixture. After 1 h, the reaction mixture wasconcentrated under reduced pressure and residual solvents were removedazeotropically by addition of toluene (5 mL) followed by concentrationunder reduced pressure (2×) to afford the title compound. This was usedin the subsequent macrolactonization without further purification.

To a suspension of 67e (78 mg, 0.13 mmol) in dichloromethane (42 mL) wasadded benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(520 mg, 0.50 mmol) and 4-dimethylaminopyridine (462 mg, 4.0 mmol) at23° C. After 24 h, the solvent was removed under reduced pressure andthe residue was purified by preparative HPLC (Gemini 5u C18 110 Åcolumn, 5-100% acetonitrile/water, 0.1% trifluoroacetic acid modifier)to afford the title compound (3.2 mg, 4%) as a white powder and as atrifluoroacetic acid salt. ¹H NMR (400 MHz, CD₃OD) δ9.65 (s, 1H), 8.69(s, 1H), 8.49 (d, J=8.7 Hz, 1H), 8.44 (d, J=8.6 Hz, 1H), 8.34 (s, 1H),7.90 (dd, J=8.7, 1.5 Hz, 1H), 7.83 (d, J=7.7 Hz, 1H), 7.78 (d, J=7.9 Hz,1H), 7.74 (s, 1H), 7.69 (app t, J=7.8 Hz, 1H), 6.82 (d, J=8.6 Hz, 1H),6.21 (q, J=6.5 Hz, 1H), 5.92-5.84 (m, 1H), 4.41-4.30 (m, 2H), 3.75 (dd,J=14.4, 6.0 Hz, 1H), 2.71 (td, J=13.0, 3.0 Hz, 1H), 2.06-1.99 (m, 1H),1.96 (dd, J=14.2, 7.1 Hz, 1H), 1.93-1.86 (m, 1H), 1.75 (s, 3H),1.73-1.59 (m, 2H), 1.68 (d, J=6.6 Hz, 3H), 1.63 (d, J=7.1 Hz, 3H), 1.47(s, 3H), 0.96 (d, J=6.8 Hz, 3H), 0.84 (d, J=6.7 Hz, 3H). HPLC Tr=5.319min. LCMS (m/z) 600.5 [M+H], Tr=2.71 min.

Examples 68 and 69

To a solution of 6-bromo-3-chloroisoquinoline (Frontier Scientific,1.594 g, 6.57 mmol) and 2,2-dimethylpropane-1,3-diol (684 mg, 6.57 mmol)in cyclopentyl methyl ether (20 mL) was added cesium carbonate (2.354 g,7.23 mmol) at 23° C. The reaction mixture was heated to 120° C. for 18h. The reaction mixture was cool to RT, diluted with ethyl acetate (50mL) and washed with water (30 mL), brine (30 mL) and the resultingorganic layer was dried over anhydrous magnesium sulfate andconcentrated under reduced pressure. The residue was purified by silicagel chromatography (80 g Isco Rf Gold Column, 0-100% ethylacetate/iso-hexanes gradient) to afford the title compound (461 mg, 23%)as a white solid.

To a solution of 68a (461 mg, 1.49 mmol) in acetonitrile (10 mL) andwater (2.5 mL) was added periodic acid (1.698 g, 7.45 mmol) at 23° C.The reaction mixture was cool to 0° C. and chromium trioxide (30 mg,0.298 mmol) was added in one portion. After 2.5 hour, the reactionmixture was diluted with ethyl acetate (30 mL) and water (30 mL) and thelayers were separated. The aqueous layer was extracted with ethylacetate (30 mL) and combined organic layers were washed with brine,dried over anhydrous magnesium sulfate and concentrated under reducedpressure to afford the title compound which was used withoutpurification (481 mg).

To a solution of 68b (481 mg, 1.49 mmol) in acetonitrile (10 mL),2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (1.133 g, 2.98 mmol),N,N-diisopropylethylamine (1.55 mL, 8.94 mmol) and (S)-methyl2-amino-3-methylbutanoate hydrochloride (749 mg, 4.47 mmol) were addedsequentially at 23° C. After 16 h, the resulting mixture wasconcentrated under reduced pressure and the residue was purifieddirectly by silica gel chromatography (24 g Isco Rf Gold Column, 0-100%ethyl acetate/iso-hexanes gradient) to afford the title compound (412mg, 63% over 2 steps) as a brown oil.

To a suspension of 68c (412 mg, 0.95 mmol) and bis(triphenylphosphine)palladium (II) dichloride in toluene (5 mL) was addedtributyl(1-ethoxyvinyl)tin (962 μL, 2.85 mmol) under argon at 23° C. Thereaction mixture was heated to 50° C. After 18 h, water (1 mL) was addedat 50° C. After 2 h, the reaction mixture was cool to RT, diluted withethyl acetate (15 mL) and 1 M solution of potassium fluoride (5 mL) wasadded. The resulting mixture was vigorously stirred at 23° C. After 2 h,the mixture was filtered through a short pad of Celite and washed withethyl acetate (10 mL). The filtrate was washed with water (15 mL), brine(15 mL), dried over anhydrous magnesium sulfate and concentrated. Thisresidue was dissolved in 1,4-dioxane (8 mL) and 1 M aqueous hydrochloricacid (1 mL) was added at 23° C. After 5 min, the reaction mixture wasquenched with a saturated solution of sodium bicarbonate (2 mL) andconcentrated to dryness. The residue was taken up in ethyl acetate (20mL) and water (20 mL) and layers were separated. The aqueous layer wasextracted with ethyl acetate (2×10 mL) and the combined organic layerswere washed with brine (20 mL), dried over anhydrous magnesium sulfateand concentrated. The residue was purified by silica gel chromatographyto afford the title compound (236 mg, 62%) as a brown gum.

Dichloro (p-cymene) ruthenium(II) dimer (2 mg, 3 μmol) and(1R,2R)-(−)-N-p-tosyl-1,2-diphenylethylenediamine (2.6 mg, 7 μmol) weresuspended in degassed water (2.5 mL) and the mixture was degassed withargon for 15 min. The mixture was stirred at 70° C. under nitrogen for90 min. The resulting yellow solution was cooled to RT. 68d (236 mg,0.59 mmol), sodium formate (200 mg, 2.95 mmol) and degassedtetrahydrofuran (1.25 mL) were added and the reaction mixture wasdegassed for 10 min. The reaction mixture was vigorously stirred at 40°C. for 3.5 h. The reaction mixture was cooled to RT and was extractedwith ethyl acetate (15 mL). The organic layer was separated, washed withwater (20 mL), brine (20 mL), dried over anhydrous magnesium sulfate,and concentrated under reduced pressure. The residue was purified bysilica gel chromatography to afford the title compound (196 mg, 83%) asa pale brown solid.

To a solution of 68e (196 mg, 0.487 mmol) in tetrahydrofuran (3 mL),methanol (1 mL) and water (1 mL) was added lithium hydroxide hydrate (23mg, 0.97 mmol) at 23° C. After 16 h, the reaction mixture wasconcentrated under reduced pressure. The residue was taken up in ethylacetate (10 mL) and water (10 mL) and acidified with 1 M aqueoushydrochloric acid solution to pH ˜2. The resulting layers wereseparated. The aqueous layer was extracted with ethyl acetate (2×10 mL)and the combined organic layers were washed with brine, dried overanhydrous magnesium sulfate and concentrated under reduced pressure toafford the title compound which was used without purification.

To a solution of(S)-1-((S)-2-tert-butoxycarbonylamino-propionyl)-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (270 mg, 0.62 mmol) in dichloromethane(5 mL) was slowly added trimethylsilyl trifluoromethanesulfonate (165μL, 0.93 mmol) at 0° C. under an argon atmosphere. After 1 hour, thereaction mixture was concentrated under reduced pressure to afford thetriflate salt of(S/R)-1-((S)-2-amino-propionyl)-hexahydro-pyridazine-3-carboxylic acid2,2,2-trichloro-ethyl ester as yellow oil which was used it withoutpurification. To a solution of 68f (189 mg, 0.49 mmol) in acetonitrile(7 mL), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (278 mg, 0.73 mmol),N,N-diisopropylethylamine (730 μL, 4.2 mmol) and(S/R)-1-((S)-2-amino-propionyl)-hexahydro-pyridazine-3-carboxylic acid2,2,2-trichloro-ethyl ester (435 mg, 0.62 mmol) in acetonitrile (3 mL)were added sequentially at 23° C. After 16 h, the resulting mixture wasconcentrated under reduced pressure and the residue was purifieddirectly by silica gel chromatography (40 g SiO₂ Isco Rf Gold Column,0-100% ethyl acetate/iso-hexanes gradient) to afford an inseparablediastereomeric mixture of the title compounds (43 mg, 13%) as acolorless residue.

To a solution of a mixture of 68g and 68h (43 mg, 61 μmol) intetrahydrofuran (4 mL) and water (2 mL) was added lithium hydroxidehydrate (3.2 mg, 0.13 mmol) at 23° C. After 1 h, the reaction mixturewas concentrated under reduced pressure and residual solvents wereremoved azeotropically by addition of toluene (5 mL) followed byconcentration under reduced pressure (2×) to afford the title compoundsas a mixture. This was used in the subsequent macrolactonization withoutfurther purification. LCMS (m/z) 572.2 [M+H], Tr=2.21 min.

To a suspension of 68i and 68j (34.8 mg, 61 μmol) in dichloromethane (20mL) was added benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate (127 mg, 0.24 mmol) and 4-dimethylaminopyridine (224mg, 1.83 mmol) at 23° C. After 24 h, the solvent was removed underreduced pressure and the residue was purified by preparative HPLC(Gemini 5u C18 110 Å column, 5-100% acetonitrile/water, 0.1%trifluoroacetic acid modifier) to afford both compounds (12 mg, 35%) asa ratio mixture of diastereomers. The diastereomers were separated usingChiral preparative HPLC to afford first eluting (Tr=3.73 min) Compound68 (2.17 mg, 6%) and the second eluting (Tr=5.713 min)

Compound 69 (1.85 mg, 5%). Compound 68: ¹H NMR (400 MHz, CD₃OD) δ8.89(s, 1H), 7.94 (d, J=8.6 Hz, 1H), 7.86 (s, 1H), 7.37 (dd, J=8.6, 1.5 Hz,1H), 7.28 (d, J=9.2 Hz, 1H), 6.93 (s, 1H), 6.09 (q, J=6.5 Hz, 1H), 5.54(q, J=7.2 Hz, 1H), 4.36-4.26 (m, 3H), 4.21 (d, J=9.7 Hz, 1H), 3.69 (dd,J=11.2, 2.6 Hz, 1H), 2.77 (td, J=12.9, 3.0 Hz, 1H), 2.00-1.84 (m, 2H),1.83-1.77 (m, 1H), 1.76-1.62 (m, 2H), 1.67 (d, J=6.7 Hz, 3H), 1.47 (d,J=7.3 Hz, 3H), 1.38 (s, 3H), 1.27 (s, 3H), 0.99 (d, J=6.7 Hz, 3H), 0.95(d, J=6.7 Hz, 3H). HPLC Tr=5.866 min. LCMS (m/z) 554.2 [M+H], Tr=2.44min.

Compound 69: ¹H NMR (400 MHz, CD3OD) δ 8.78 (s, 1H), 7.83 (d, J=8.6 Hz,1H), 7.74 (s, 1H), 7.24 (dd, J=8.6, 1.6 Hz, 1H), 7.03 (s, 1H), 5.93 (q,J=6.7 Hz, 1H), 5.35 (q, J=6.8 Hz, 1H), 4.31 (d, J=9.8 Hz, 1H), 4.18 (d,J=13.3 Hz, 1H), 4.02-3.97 (m, 2H), (d, J=10.0 Hz, 1H), 3.65 (dd, J=10.8,2.8 Hz, 1H), 2.71 (td, J=12.7, 2.8 Hz, 1H), 1.98-1.89 (m, 1H), 1.87-1.77(m, 2H), 1.73-1.64 (m, 1H), 1.57 (d, J=6.7 Hz, 3H), 1.49 (d, J=7.0 Hz,3H), 1.40 (s, 3H), 1.13 (s, 3H), 0.89 (d, J=6.7 Hz, 3H), 0.85 (d, J=6.5Hz, 3H) HPLC Tr=5.951 min. LCMS (m/z) 555.2 [M+H], Tr=2.49 min.

Example 70

To 2,7-dibromonaphthalene (1 g, 3.50 mmol) in anhydrous tetrahydrofuran(18 mL), at −78° C. and under an atmosphere of nitrogen, was added asolution of n-butyllithium (2.5 M in hexanes, 1.5 mL, 3.67 mmol)dropwise. The reaction was stirred at −78° C. for 20 min after whichN-methoxy-N-methylacetamide (409 μL, 3.85 mmol) was added. After 15 min,the reaction was warmed to RT and stirred for 30 min. The reaction wasquenched with 2 M hydrochloric acid and extracted twice withdichloromethane. The combined organic layers were dried through ahydrophobic frit and concentrated in vacuo. The product was purified bysilica flash chromatography (iso-hexanes/ethyl acetate, 7/1) to affordthe title compound (650 mg, 75%) as a colorless solid.

To dichloro (p-cymene) ruthenium (II) dimer (8 mg, 0.013 mmol) in water(5 mL) at RT was added (1R,2R)-(−)-N-p-tosyl-1,2-diphenylethylenediamine(11.5 mg, 0.031 mmol). The system was degassed for 15 min and thenheated to 70° C. for 1.5 h. The reaction was cooled and was added asolution of 70a (650 mg, 2.61 mmol) in degassed anhydroustetrahydrofuran (2 mL) followed by sodium formate (874 mg, 13.1 mmol).The reaction was heated at 40° C. for 3 h, cooled to RT and extractedtwice with dichloromethane. The combined organic layers were driedthrough a hydrophobic frit and concentrated in vacuo. The product waspurified by silica gel chromatography (iso-hexanes/ethyl acetate, 4/1)to afford the title compound (450 mg, 69%) as a colorless solid.

70b (42 mg, 0.17 mmol) was dissolved in acetonitrile (2 mL) in amicrowave vial, to the mixture was added 3-butenoic acid (35 mg, 0.41mmol), palladium (II) acetate (4 mg, 0.017 mmol), tri-(o-tolyl)phosphine(10 mg, 0.034 mmol) and triethylamine (0.12 mL). The vial was heated at120° in the microwave reactor for 15 mins. The reaction mixture was thenfiltered, the solvent was evaporated and purified with combi-flashcolumn chromatography (Eluent methanol/dichloromethane 1:3) to affordthe title compound (35 mg, 81%) as a yellow solid.

70c (40 mg, 0.08 mmol) was dissolved in N,N-dimethylformamide (2 mL), tothe solution was added 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methanaminium (36 mg, 0.1 mmol), thereaction mixture was stirred at RT for 10 mins. Then(S)-1-[(S)-2-((S)-2-amino-3-methylbutyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester (0.18 mmol) in N,N-dimethylformamide (1mL) was added to above reaction mixture followed by triethylamine (32mg, 0.32 mmol). The reaction mixture was stirred at RT for 1 h, and thenit was diluted with ethyl acetate (20 mL) and washed with brine. Theaqueous layer was back extracted with ethyl acetate and the combinedorganic solvent was evaporated and purified with combi-flash columnchromatography (Eluent methanol/dichloromethane 1:10) to afford thetitle compound (68 mg, 65%) as a yellow solid.

70d (55 mg, 0.08 mmol) was dissolved in the mixture of tetrahydrofuran(2 mL), methanol (1 mL) and water (1 mL). To the solution was addedlithium hydroxide hydrate (4 mg, 0.16 mmol). The reaction mixture wasstirred at RT for 1 h. Dichloromethane (10 mL) and water (10 mL) wereadded to the reaction mixture. 1 N Hydrochloric acid was added to theaqueous layer until pH reached 2, the acidic aqueous layer was extractedwith dichloromethane (2×10 mL). The organic solvent was then evaporatedto afford the title compound (37 mg, 84%) as a white solid.

2-Methyl-6-nitrobenzoic anhydride (19 mg, 0.056 mmol) and4-dimethylaminopyridine (23 mg, 0.185 mmol) were dissolved in1,2-dichloroethane (18 mL) and the solution was heated at 50° C. To theabove solution was added 70e (20 mg, 0.037 mmol) inN,N-dimethylformamide (1 mL) via syringe pump in 10 h. The reactionmixture was stirred at 50° C. for 2 h after the completion of addition.The solvent was then evaporated and the residue was purified by reversephase preparative HPLC (0-100% acetonitrile/water) to afford the titlecompound (4 mg, 22%) as a white solid. ¹H NMR (400 MHz, CD₃OD)δ7.79-7.72 (m, 3H), 7.52 (dd, J=1.6, 8.8 Hz, 1H), 7.41 (s, 1H), 7.33(dd, J=8.4, 1.6 Hz, 1H), 6.48 (d, J=16.4 Hz, 1H), 6.31-6.24 (m, 1H),6.02 (q, J=7.6 Hz, 1H), 4.64 (d, J=12.4 Hz, 1H), 4.40 (d, J=12.8 Hz,1H), 4.30 (d, J=10.4 Hz, 1H), 3.78-3.72 (m, 1H), 3.36-3.32 (m, 2H),2.99-2.93 (m, 1H), 2.77-2.71 (m, 1H), 1.99-1.69 (m, 5H), 1.66 (d, J=6.8Hz, 3H), 1.58 (d, J=7.2 Hz, 3H), 0.99 (d, J=6.8 Hz, 6H). LCMS (m/z)521.1 [M−H], Tr=3.18 min.

Example 71

Compound 70 (5 mg, 0.01 mmol) was dissolved in ethanol (5 mL) underargon, to the solution was added Pd (10% on activated carbon, 3 mg). Thereaction flask was then purged and then charged with H₂ using a balloon.The reaction was filtered through Celite after 2 h, the filtrate wasevaporated under reduced pressure and purified by reverse phasepreparative HPLC (0-100% acetonitrile/water) to afford the titlecompound (3.2 mg, 64%) as a white powder. ¹H-NMR (400 MHz, CD₃OD) δ8.47(d, J=6.0 Hz, 1H), 7.97 (d, J=8.8 Hz, 1H), 7.69-7.62 (m, 3H), 7.31 (s,1H), 7.24-7.19 (m, 2H), 5.97 (dd, J=12.8, 6.0 Hz, 1H), 5.62 (pent, J=7.2Hz, 1H), 4.76-4.16 (m, 1H), 4.10 (app t, J=12.8 Hz, 1H), 4.05-3.69 (m,1H), 2.85 (br s, 1H), 2.73-2.41 (m, 2H), 2.40-2.37 (m, 1H), 2.10-1.93(m, 1H), 1.92-1.70 (m, 6H), 1.53 (d, J=6.8 Hz, 3H), 1.49 (d, J=7.6 Hz,3H), 0.99 (d, J=6.8 Hz, 6H). LCMS (m/z) 523.140 [M+H], Tr=3.16 min.

Example 72

Into an oven dried, argon purged flask Compound 49 (10 mg, 0.017 mmol),copper(I) iodide (1 mg, 0.005 mmol) and prop-2-yn-1-ol (4 mg, 0.07 mmol)were added. The flask was sealed and repurged with argon three times.Anhydrous N,N-dimethylformamide (5 mL) was added and the reactionmixture was repurged with argon three times. This reaction mixture wasstirred at RT for 12 h. After evaporation of the solvent under reducedpressure, the crude residue was dissolved in ethyl acetate (10 mL) andfiltered through filter aid and the filter pad was washed with ethylacetate (10 mL). After concentration under reduced pressure, the residuewas purified by silica gel chromatography (gradient from 0-40% ethylacetate and methanol (4/1) in iso-hexanes) to afford the title compound(10 mg, 93%) as a white solid after evaporation. R_(f)=0.33, 10%methanol in dichloromethane. ¹H NMR (400 MHz, CD₃OD) δ9.05 (s, 1H), 7.95(d, J=8.6 Hz, 1H), 7.94 (s, 1H), 7.82 (s, 1H), 7.50 (s, 1H), 7.46 (d,J=8.6 Hz, 1H), 6.49 (d, J=16.0 Hz, 1H), 6.40 (d, J=16.0 Hz, 1H), 5.95(m, 1H), 5.50 (m, 1H), 4.59-4.53 (m, 3H), 4.36 (m, 2H), 4.30 (m, 1H),3.68 (m, 1H), 2.80 (m, 1H), 2.60 (m, 1H), 2.23 (m, 1H), 2.14 (m, 1H),1.90-1.58 (m, 4H), 1.57 (d, J=6.6 Hz, 3H), 1.55 (d, J=7.6 Hz, 3H), 1.41(s, 3H), 1.27 (s, 3H). LCMS (m/z) 633.4 [M+H], Tr=2.66 min.

Example 73

To 3-Nitromethylene-oxetane (Angew. Chem. Int. Ed. 2006, 45 (46), 7736,2.5 g, 21.7 mmol) in anhydrous tetrahydrofuran (40 mL) at RT and underan atmosphere of nitrogen was added copper(I) iodide (413 mg, 2.17 mmol)and chlorotrimethylsilane (3.0 mL, 23.9 mmol). The resulting yellowsolution was stirred for 5 min and cooled to between −15° C. and −11° C.with a methanol ice bath. Vinyl magnesium bromide (43.5 mL, 43.5 mmol,1.0 M in tetrahydrofuran) was slowly added over 3 h via a syringe pump.Following the addition the reaction was quenched with a saturatedaqueous solution of ammonium chloride, filtered and extracted withdiethyl ether (3×). The combined organics were dried through ahydrophobic frit and concentrated in vacuo. The residue was purified bysilica gel chromatography using diethyl ether to give the title compoundas a yellow oil (2.0 g, 64%).

To 73a (600 mg, 4.2 mmol) in anhydrous dimethylsulfoxide (13 mL) wasadded acetic acid (2.4 mL, 42 mmol) and sodium nitrite (869 mg, 12.6mmol) and the mixture heated to 35° C. for 16 h. The reaction was cooledto RT and diluted with water. The pH was adjusted to pH 3-4 with 10%aqueous hydrochloric acid and the product extracted with diethyl ether(3×) and diethyl ether/ethyl acetate (1:1). The combined organics weredried through a hydrophobic frit and concentrated in vacuo. The residuewas purified by silica gel chromatography using iso-hexanes/ethylacetate (2/1 then 1/1) to afford impure acid. This was dissolved indiethyl ether and extracted with a saturated solution of sodiumcarbonate. The aqueous was acidified to pH 2 with concentratedhydrochloric acid and extracted with ethyl acetate (3×). The organicswere dried through a hydrophobic frit and concentrated in vacuo to givethe title compound as a yellow oil (228 mg, 43%).

To 73b (150 mg, 1.18 mmol) in anhydrous dioxane (1 mL), was added aceticacid (R)-1-(7-bromo-quinolin-2-yl)-ethyl ester (347 mg, 1.18 mmol)followed by dicyclohexylmethylamine (0.76 mL, 3.54 mmol), palladium(II)acetate (53 mg, 0.24 mmol) and Tri(o-tolyl) phosphine (72 mg, 0.24mmol). The mixture was heated at 100° C. for 1 hour. An additionalamount of palladium(II) acetate (26 mg, 0.12 mmol) and Tri(o-tolyl)phosphine (36 mg, 0.12 mmol) was added and heating continued at 100° C.for a further 45 min. The reaction was cooled to RT and 2M HCl addeduntil pH 3-4 was reached. The product was extracted with ethyl acetate(3×) and ethyl acetate/10% methanol (2×). The combined organics weredried through a hydrophobic frit and concentrated in vacuo to yield thetitle compound as a brown oil.

A solution of 1e (3.01 g, 5.66 mmol) in tetrahydrofuran:methanol (1:1,60 mL) was stirred at 0° C. Tetra-n-butylammonium fluoride (1 M intetrahydrofuran, 11.3 mL, 11.3 mmol) was added and the reaction mixturewas stirred at RT for 22 h. The solvent was evaporated and the residuewas purified by silica gel chromatography using iso-hexane toiso-hexane/ethyl acetate 1:1 afford the title compound (2.14 g, 91%) asa white foam.

To 73d (365 mg, 0.88 mmol) in anhydrous dichloromethane (15 mL) at 0° C.and under an atmosphere of nitrogen, was added trimethylsilyltrifluoromethanesulfonate (239 μL, 1.32 mmol). The reaction mixture wasstirred at 0° C. for 1 hour before adding N,N-diisopropylethylamine (613μL, 3.52 mmol) and then concentrated in vacuo, and co-evaporated withtoluene to afford(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester as a white solid. To 73c (300 mg, 0.88mmol) in anhydrous acetonitrile (9 mL) at 0° C. and under an atmosphereof nitrogen was added N,N-diisopropylethylamine (766 μL, 4.4 mmol) and2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (401 mg, 1.06 mmol). The solution wasstirred at 0° C. for 3 min before adding a solution of(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid 2,2,2-trichloro-ethyl ester in anhydrous acetonitrile (2 mL). Thereaction was warmed to RT stirred for 2 h. The reaction was quenchedwith 1M HCl and extracted with ethyl acetate (3×). The combined organiclayers were dried through a hydrophobic frit and concentrated in vacuo.The residue was purified by silica gel chromatography using ethylacetate to give the title compound as a viscous yellow oil (160 mg, 22%,2 steps).

To 73e (160 mg, 0.25 mmol) in tetrahydrofuran (10 mL) and water (2 mL)was added lithium hydroxide monohydrate (53 mg, 1.25 mmol) at 0° C. Thereaction was stirred at 0° C. for 2 h and quenched by adding 2M aqueoushydrochloric acid (0.63 mL). The reaction was concentrated in vacuo,followed by co-evaporation from toluene/methanol (3×) and then toluene(3×) and dried on a high vacuum for 15 min. The resulting residue wasdissolved in anhydrous tetrahydrofuran (83 mL) and at RT was addedN,N-diisopropylethylamine (223 μL, 1.25 mmol),2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (114 mg, 1.3 mmol) and4-dimethylaminopyridine (3 mg, 0.03 mmol). The reaction was stirred for24 h, diluted with ethyl acetate and washed with 1M HCl (1×) and brine(1×). The organic layer was dried through a hydrophobic frit andconcentrated in vacuo. The residue was purified by silica gelchromatography using ethyl acetate/methanol 1:0 then 20/1 to give aviscous yellow oil (52 mg). This was further purified by preparativethin layer chromatography (ethyl acetate) to afford the title compoundas a white solid which was triturated with diethyl ether, filtered andvacuum dried. (22 mg, 16%, 2 steps). ¹H NMR (300 MHz, CDCl₃) 0.96 (d,J=6.5 Hz, 3H), 1.00 (d, J=6.5 Hz, 3H), 1.50 (d, J=7.1 Hz, 3H), 1.69 (d,J=6.9 Hz, 3H), 1.81-2.16 (m, 4H), 2.63-2.74 (m, 1H), 3.26-3.38 (m 1H),3.70-3.83 (m, 1H), 3.98 (d, J=12.3 Hz, 1H), 4.28 (t, J=9.8 Hz, 1H),4.33-4.43 (m, 1H), 4.57 (d, J=5.8 Hz, 1H), 4.86 (q, J=6.7 Hz, 2H), 5.13(d, J=5.8 Hz, 1H), 5.73 (t, J=6.9 Hz, 1H), 5.96 (q, J=6.7 Hz, 1H), 6.24(d, J=16.3 Hz, 1H), 6.61-6.73 (m, 1H), 7.00 (d, J=16.3 Hz, 1H),7.09-7.20 (m, 1H), 7.40 (d, J=8.5 Hz, 1H), 7.58 (s, 1H), 7.81-7.93 (m,2H), 8.22 (d, J=8.7 Hz, 1H). LCMS (m/z)=564.2 [M+H], Tr=1.96 min.

Example 74

Compound 57 (20 mg, 0.036 mmol) was dissolved in methanol (5 mL) andadded catalytic amount of 10% Pd on carbon. The reaction mixture wasstirred under atmosphere hydrogen for 2 h. The catalyst was removed byfiltration and the eluent was concentrated under reduced pressure.Purification by reverse phase preparative HPLC gave the title compound(8.4 mg, 42% yield) as a white powder. ¹H NMR (300 MHz, CD₃OD): δ9.61(s, 1H), 8.42 (d, J=9.0 Hz, 1H), 7.99 (s, 1H), 7.89 (d, J=9.0 Hz, 1H),7.83 (br s, 1H), 7.29 (d, J=8.7 Hz, 1H), 6.27-6.21 (m, 1H), 5.92-5.82(m, 1H), 3.96-3.5 (m, 4H), 3.28-3.19 (m, 1H), 3.08-2.98 (m, 1H),2.30-2.20 (m, 1H), 2.10-1.90 (m, 4H), 1.78-1.72 (m, 5H), 1.56-1.51 (m,6H), 1.21 (s, 3H), 0.89 (d, J=7.2 Hz, 6H). LCMS (m/z): 552.3 [M+H],Tr=1.74 min.

Example 75

A solution of ethyl acetoacetate (20 g, 19.4 mL, 0.154 mol) in a mixtureof dioxane (120 mL) and aqueous formaldehyde (37% solution in water,57.7 mL, 0.77 mol) was stirred at RT. Triethylamine (1.0 M intetrahydrofuran, 7.7 mL, 7.7 mmol) was added and the reaction mixturewas heated at 60° C. for 20 h and then heated at 100° C. for 4 h. Thereaction mixture was cooled to RT and was poured into water (1500 mL).The aqueous solution was washed with toluene. The aqueous layer wasconcentrated to ˜50% of the initial volume and extracted with ethylacetate. The organic extracts were combined and evaporated. The residuewas purified by silica gel chromatography using a gradient of iso-hexaneto iso-hexane/ethyl acetate 1:1 followed by silica gel chromatographyusing a gradient of iso-hexane to iso-hexane/ethyl acetate 3:2 to affordthe title compound (2.33 g, 8%) as a yellow oil.

A solution of 75a (2.10 g, 11 mmol), 2,2-dimethoxypropane (13.5 mL, 110mmol) and 4-toluenesulfonic acid hydrate (209 mg, 1.1 mmol) in acetone(8 mL) was stirred at RT for 18 h. Saturated sodium hydrogen carbonatesolution was added and the mixture was extracted with ethyl acetate. Theorganic extracts were combined, washed with water and brine. The organiclayer was filtered through a hydrophobic frit and the filtrate wasevaporated. The residue was purified by silica gel chromatography usinga gradient of iso-hexane/ethyl acetate 1:5 to 3:7 to afford the titlecompound (1.88 g, 74%) as a colorless oil.

A solution of N,N-diisopropylethylamine (0.55 mL, 3.9 mmol) intetrahydrofuran (20 mL) was stirred at −78° C. under nitrogen. n-Butyllithium (1.6 M in hexane, 2.25 mL, 3.6 mmol) was added dropwise and thereaction mixture was warmed to 0° C. The reaction mixture was stirred at0° C. for 5 min and then cooled to −78° C. A solution of 75b (690 mg,3.0 mmol) in tetrahydrofuran (3 mL) was added dropwise and the reactionmixture was stirred at −78° C. for 15 min. A solution ofN-phenyl-(bistrifluoromethanesulfonamide) (1.18 g, 3.3 mmol) intetrahydrofuran (10 mL) was added dropwise over 5 min and the reactionmixture was stirred at −78° C. for 15 min. The cooling bath was removedand the reaction mixture was warmed to RT and then stirred at RT for 90min. The solvent was evaporated and diethyl ether (30 mL) was added. Thesolution was cooled to 5° C. and was washed with cold 1 M sodiumhydroxide solution (3×30 mL). The organic layer was separated, washedwith brine, dried over anhydrous sodium sulfate, filtered and thesolvent evaporated to afford the title compound (960 mg, 88%) as ayellow oil which was used directly in the next step.

A solution of 75c (470 mg, 1.3 mmol) and tri-n-butylamine (721 mg, 0.93mL, 3.9 mmol) in N,N-dimethylformamide (3 mL) was stirred at RT undernitrogen. Bis(triphenylphosphine)palladium(II) dichloride (45 mg, 0.065mmol) and formic acid (120 mg, 0.1 mL, 2.6 mmol) was added and thereaction mixture was heated at 60° C. for 90 min. The reaction mixturewas cooled to RT and ethyl acetate and water was added. The organicextract was separated, washed with water (×5), and brine. The organicsolution was filtered through a hydrophobic frit and the filtrate wasevaporated. The residue was purified by silica gel chromatography usingiso-hexane/ethyl acetate 1:9 to afford the title compound (1.88 g, 74%)as a colorless oil.

A solution of 75d (150 mg, 0.7 mmol) in tetrahydrofuran (3 mL) wasstirred at 5° C. under nitrogen. A solution of lithium hydroxidemonohydrate (59 mg, 1.4 mmol) in water (1 mL) was added and the reactionmixture was stirred at 5° C. for 30 min and then at RT for 5 h. Methanol(0.5 mL) was added to give a clear solution and the reaction mixture wasstirred at RT for 22 h. The solvent was evaporated. Water (2 mL) wasadded to the residue and the solution was acidified to pH 2 with 2 Mhydrochloric acid. Brine was added and the mixture was extracted withethyl acetate. The organic extracts were combined, washed with brine.The organic extract was separated, washed with water (×5), and brine.The organic solution was filtered through a hydrophobic frit and thefiltrate was evaporated to afford the title compound (117 mg, 90%) as acolorless oil.

A mixture of 75e (96 mg, 0.5 mmol) and acetic acid(R)-1-(7-bromo-quinolin-2-yl)-ethyl ester (147 mg, 0.5 mmol) inacetonitrile (4 mL) was stirred at RT. Tri(o-tolyl)phosphine (46 mg,0.15 mmol), palladium(II) acetate (17 mg, 0.075 mmol) and triethylamine(101 mg, 0.14 mL, 1.0 mmol) was added and the reaction mixture washeated in a microwave reactor at 100° C. for 20 min. The solvent wasevaporated. Water and ethyl acetate was added and the mixture wasacidified to pH 3-4 with 2 M hydrochloric acid. The mixture wasextracted with ethyl acetate and the organic extracts were combined andwashed with brine. The organic solution was filtered through ahydrophobic frit and the filtrate was evaporated. The residue waspurified by silica gel chromatography using iso-hexane/ethyl acetate 1:1to ethyl acetate to ethyl acetate/methanol 5:1. The residue wasco-evaporated with ethyl acetate and then dichloromethane and dried toafford the title compound (134 mg, 67%) as a yellow gum.

A solution of(S)-1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid methyl ester (150 mg, 0.36 mmol) in dichloromethane (5 mL) wasstirred at 0° C. under nitrogen. Trimethylsilyltrifluoromethanesulfonate (160 mg, 0.13 mL, 0.72 mmol) was added and thereaction mixture was stirred at 0° C. for 1 hour.N,N-Diisopropylethylamine (186 mg, 0.25 mL, 1.44 mmol) was added and thesolvent was evaporated to afford(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid methyl ester (0.36 mmol) as a white solid which was used withoutfurther purification. A solution of 75f (134 mg, 0.33 mmol) inacetonitrile was stirred at 0° C. under nitrogen. A solution of(S)-1-[(S)-2-((S)-2-amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylicacid methyl ester (0.36 mmol) and N,N-diisopropylethylamine (129 mg,0.17 mL, 1.0 mmol) in acetonitrile (5 mL) was added followed by2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (175 mg, 0.46 mmol) and the reactionmixture was stirred at 0° C. for 10 min and then at RT for 1 hour. Thesolvent was evaporated and the residue was partioned between ethylacetate and water. The organic extracts were combined and washed withbrine. The organic solution was filtered through a hydrophobic frit andthe filtrate was evaporated. The residue was purified by silica gelchromatography using iso-hexane/ethyl acetate 1:5 to ethyl acetate toafford the title compound (165 mg, 72%) as a yellow solid.

A solution of 75g (35 mg, 0.05 mmol) in tetrahydrofuran (0.5 mL) wasstirred at 5° C. under nitrogen. A solution of lithium hydroxidemonohydrate (8.4 mg, 0.2 mmol) in water (0.5 mL) was added followed bymethanol (0.5 mL) and the reaction mixture was stirred at 5° C. for 1hour. 1 M hydrochloric acid (0.2 mL) was added and the solvent wasevaporated. The residue was co-evaporated with methanol/toluene (1:1,×2) followed by toluene (×2). The residue was triturated with diethylether (×3) and dried to afford the title compound (0.05 mmol) as a paleyellow solid which was used crude in the next reaction.

A solution of 2-methyl-6-nitrobenzoic anhydride (86 mg, 0.25 mmol) and4-dimethylaminopyridine (46 mg, 0.38 mmol) in 1,2-dichloroethane (16 mL)was stirred at RT under nitrogen. A molecular sieves (400 mg) was addedand the suspension was heated at 50° C. A solution of crude 75h (0.05mmol) in N,N-dimethylformamide (2 mL) was added dropwise over 4 h andthe reaction mixture was stirred at 50° C. for an additional 1 hour. Thereaction mixture was cooled to RT and the mixture was filtered throughcelite. The filter pad was washed with ethyl acetate and the filtratewas evaporated. The residue was diluted with ethyl acetate and thesolution was washed with brine (×3). The organic layer was separated andevaporated. The organic solution was filtered through a hydrophobic fritand the filtrate was evaporated. The residue was purified by preparativeHPLC to afford the title compound (4.2 mg, 13%) as a white solid. ¹H NMR(300 MHz, CD₃OD) 1.00 (d, J=6.7 Hz, 3H), 1.01 (d, J=6.6 Hz, 3H), 1.37(s, 3H), 1.47 (s, 3H), 1.63 (d, J=7.1 Hz, 3H), 1.64-1.70 (m, 2H), 1.73(d, J=6.9 Hz, 3H), 1.85-2.10 (m, 3H), 2.70-2.80 (m, 1H), 3.78-3.87 (m,1H), 3.87 (d, J=11.1 Hz, 1H), 4.03-4.07 (m, 1H), 4.26-4.46 (m, 5H), 5.72(q, J=7.1 Hz, 1H), 5.93 (q, J=6.8 Hz, 1H), 6.38 (s, 2H), 7.42 (d, J=8.5Hz, 1H), 7.61 (br s, 1H), 7.78-7.86 (m, 2H), 8.22 (d, J=8.5 Hz, 1H).LCMS (m/z) 622.2 [M+H], Tr=2.24 min.

Example 76

A suspension of crude 75h (200 mg, 0.25 mmol) in tetrahydrofuran (100mL) was stirred at RT under nitrogen. N,N-diisopropylethylamine (161 mg,0.22 mL, 1.25 mmol) and 4-dimethylaminopyridine (15 mg, 0.125 mmol) wasadded and the suspension was stirred for 5 min.2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (133 mg, 0.35 mmol) was added and thereaction mixture was stirred at RT for 2 h. Additional2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (57 mg, 0.15 mmol) was added and thereaction mixture was stirred at RT for 1 hour. The solvent wasevaporated and the residue was diluted with ethyl acetate and 2 Mhydrochloric acid. The organic solution was separated and washed withwater, saturated sodium hydrogen carbonate solution, and brine. Theorganic solution was filtered through a hydrophobic frit and thefiltrate was evaporated. The residue was purified by silica gelchromatography using iso-hexane/ethyl acetate 7:3 to ethyl acetate toethyl acetate/methanol 9:1. The residue was purified by preparative HPLCto afford the title compound (20 mg, 14%) as a white solid. ¹H NMR (500MHz, CD₃OD) 1.00 (d, J=6.5 Hz, 3H), 1.01 (d, J=6.5 Hz, 3H), 1.68 (d,J=7.1 Hz, 3H), 1.73 (d, J=6.7 Hz, 3H), 1.74-2.05 (m, 5H), 2.72-2.80 (m,1H), 3.70 (d, J=11.1 Hz, 1H), 3.81-4.07 (m, 4H), 4.35-4.47 (m, 3H), 5.77(q, J=7.1 Hz, 1H), 5.92 (q, J=6.8 Hz, 1H), 6.43 (s, 2H), 7.40 (d, J=8.5Hz, 1H), 7.63 (s, 1H), 7.82 (s, 2H), 8.21 (d, J=8.5 Hz, 1H). LCMS (m/z)582.2 [M+H], Tr=1.66 min.

Biological Examples: Inhibition of Peptidyl-Prolyl Isomerase (PPlase)Activity

The PPlase assay was based on the procedure reported by Janowski et al.(Anal. Biochem. 1997, 252, 299). Assay buffer (1980 L of a solutioncontaining 35 mM HEPES pH 7.8, 50M DTT, and 0.01% N P40) waspre-equilibrated to 10° C. in a quartz cuvette equipped with an overheadstirrer. To this solution was added 10 L of compound in DMSO (finalconcentration: 0.5% DMSO), followed by 5 L of a 2M stock solution ofcyclophilin A (final concentration: 5 nM). The reaction was initiatedwith the addition of 5 L of 40 mM of the tetrapeptide Succ-AAPF-pNA(100M final concentration) dissolved in a solution of 0.5M LiCl intrifluoroethanol. Upon the initiation of the reaction, the absorbance ofthe peptide substrate was monitored at 330 nm for five minutes using aBeckman Coulter DU800 spectrophotometer. Progress curves were fit with asingle-exponential decay model to calculate rates. The IC₅₀ values werecalculated with a four-parameter logistic fit using GraphPad Prismsoftware.

Cyclophilin A TR-FRET Competitive Binding Assay

Inhibitor potency was measured using a competitive binding assay with atime-resolved fluorescence resonance energy transfer (TR-FRET) readout.To a reaction buffer consisting of 35 mM HEPES pH 7.8, 100 mM NaCl,0.01% NP-40 (Pierce), 1 mM DTT, and 1% DMSO were added the following: 5nM of cyclophilin A modified at the N-terminus with an 8× histidineaffinity tag (CypA); 150 nM of cyclosporin A modified with a linkerattached to a Cy5 fluorophore (CsA-Cy5); 1 nM Eu-labeled anti-(6×His)antibody (Perkin-Elmer); and test compound at one of variousconcentrations. The total volume of the assay solution was 100 L. Aftera two-hour incubation, the TR-FRET was measured using a Perkin ElmerEnvision plate reader (excitation at 340 nm, emission measured at 590 nmand 665 nm). The signal was calculated as the ratio of the emission at665 nm to that at 590 nm. An IC₅₀ value was calculated using afour-parameter logistic fit.

When tested, certain compounds of this invention were found to inhibitcyclophilin binding as listed in Table 1 below. The IC₅₀'s are presentedas ranges wherein A is 100 nM, B is 101 to 1000 nM and C is 1001 to10,000 nM.

Antiviral Activity

The antiviral activity of a compound can be measured using standardscreening protocols: for example, cell-based Flavivirus immunodetectionassay and cell-based Flavivirus cytopathic effect assay as described inU.S. Patent Publication Number US/20130022573, which is herebyincorporated by reference in its entirety.

One aspect of the invention relates to methods of inhibiting viralinfections, comprising the step of treating a sample or subjectsuspected of needing such inhibition with a composition of theinvention. The antiviral activity of a compound of the invention can bemeasured using standard screening protocols that are known.

The anti-HCV activity of the compounds of this invention was tested in ahuman hepatoma Huh-7 cell line harboring a HCV replicon. The assaycomprised the following steps:

Step 1 (Compound Preparation and Serial Dilution):

Serial dilution was performed in 100% DMSO in a 384-well plate. Asolution containing a compound at 225-fold concentration of the startingfinal serial dilution concentration was prepared in 100% DMSO and 15 μLadded to the pre-specified wells in column 3 or 13 of a polypropylene384-well plate. The rest of the 384-well plate was filled with 10 μL100% DMSO except for columns 23 and 24, where 10 μL of 500 μM a HCVprotease inhibitor (ITMN-191) in 100% DMSO was added. The HCV proteaseinhibitor was used a control of 100% inhibition of HCV replication. Theplate was then placed on a Biomek FX Workstation to start the serialdilution. The serial dilution was performed for ten cycles of 3-folddilution from column 3 to 12 or from column 13 to 22.

Step 2 (Cell Culture Plate Preparation and Compound Addition):

To each well of a black polypropylene 384-well plate, 90 μL of cellmedia containing 1600 suspended Huh-7 HCV replicon cells was added witha Biotek uFlow Workstation. A volume of 0.4 μL of the compound solutionwas transferred from the serial dilution plate to the cell culture plateon a Biomek FX Workstation. The DMSO concentration in the final assaycondition was 0.44%. The plates were incubated for 3 days at 37° C. with5% CO₂ and 85% humidity.

Step 3 (Detection of Cytotoxicity and Inhibition of Viral Replication):

a) Assessment of cytotoxicity: The media in the 384-well cell cultureplate was aspirated with a Biotek EL405 plate-washer. A volume of 50 μLof a solution containing 400 nM Calcein AM in 100% PBS was added to eachwell of the plate with a Biotek uFlow Workstation. The plate wasincubated for 30 minutes at RT before the fluorescence signal (emission490 nm, exitation 520 nm) was measured with a Perkin Elmer EnvisionPlate Reader.

b) Assessment of inhibition of viral replication: The calcein-PBSsolution in the 384-well cell culture plate was aspirated with a BiotekEL405 plate-washer. A volume of 20 μL of Dual-Glo luciferase buffer(Promega, Dual-Glo Luciferase Assay Reagent, cat. #E298B) was added toeach well of the plate with a Biotek uFlow Workstation. The plate wasincubated for 10 minutes at RT. A volume of 20 μL of a solutioncontaining 1:100 mixture of Dual-Glo Stop & Glo substrate (Promega,Dual-Glo Luciferase Assay Reagent, cat. #E313B) and Dual-Glo Stop & Globuffer (Promega, Dual-Glo Luciferase Assay Reagent, cat. #E314B) wasthen added to each well of the plate with a Biotek uFlow Workstation.The plate was incubated at RT for 10 minutes before the luminescencesignal was measured with a Perkin Elmer Envision Plate Reader.

Step 4 (Calculation):

The percent cytotoxicity was determined by calcein AM conversion tofluorescent product. The average fluorescent signal from the DMSOcontrol wells were defined as 100% nontoxic. The individual fluorescentsignal from testing compound treated well was divided by the averagesignal from DMSO control wells and then multiplied by 100% to get thepercent viability. The percent anti-HCV replication activity wasdetermined by the luminescence signal from the testing well compared toDMSO controls wells. The background signal was determined by the averageluminescence signal from the HCV protease inhibitor treated wells andwas subtracted from the signal from the testing wells as well as theDMSO control wells. Following 3-fold serial dilutions, the EC₅₀ and CC₅₀values were calculated by fitting % inhibition at each concentration tothe following equation:

% inhibition=100%/[(EC₅₀/[I])^(b)+1]

where b is Hill's coefficient. See, for reference, Hill, A. V., ThePossible Effects of the Aggregation of the Molecules of Hæmoglobin onits Dissociation Curves, J. Physiol. 40: iv-vii. (1910). % inhibitionvalues at a specific concentration, for example 2 μM, can also bederived from the formula above.

When tested, certain compounds of this invention were found to inhibitviral replication as listed in Table 1. The EC₅₀'s are presented as a %inhibition.

TABLE 1 Example TR- Replicon 1a No. FRET % inhibition at 1 μm 1 A 32 2 B63 3 A 44 4 B 58 5 A 80 6 A 86 7 A 94 8 B 37 9 A 98 10 B 28 11 A 6 12 C0 13 A 87 14 A 94 15 A 99 16 B 94 17 A 100 18 A 94 19 A 97 20 A 99 21 A93 22 A 100 23 A 99 24 A 99 25 B 71 26 A 99 27 A 100 28 B 56 29 C 28 30A 73 31 A 98 32 A 71 33 C 14 34 A 92 35 A 94 36 A 73 37 A 76 38 A 98 39A 98 40 A 96 41 A 96 42 B 16 43 A 97 44 A 78 45 A 80 46 A 90 47 B 9 48 A40 49 A 85 50 B 12 51 A 83 52 A 98 53 B 89 54 A 98 55 B 76 56 B 47 57 A100 58 A 99 59 A 100 60 B 65 61 A 72 62 B 25 63 B 80 64 B 35 65 A 92 66A 80 67 A 97 68 A 91 69 C 19 70 A 95 71 B — 72 B 0 73 A 94 74 A 95 75 A100 76 A 63

The specific pharmacological and biochemical responses observed may varyaccording to and depending on the particular active compound selected orwhether there are present pharmaceutical carriers, as well as the typeof formulation and mode of administration employed, and such expectedvariations or differences in the results are contemplated in accordancewith practice of the present invention.

Although specific embodiments of the present invention are hereinillustrated and described in detail, the invention is not limitedthereto. The above detailed descriptions are provided as exemplary ofthe present invention and should not be construed as constituting anylimitation of the invention.

1. A compound of Formula I:

or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture ofstereoisomers, tautomer, ester or prodrug thereof, wherein: A¹ is(C₂-C₅)alkylene, (C₂-C₅)alkenylene, (C₂-C₅)alkynylene,—O—(C₂-C₄)alkylene, —O—(C₂-C₄)alkenylene, arylene, aryl(C₁-C₂)alkylene,heterocycloalkylene, pyrazolylene, pyridylene, pyrimidinylene orheterocycloalkyl(C₁-C₂)alkylene, wherein a sp³ carbon atom of A¹ isoptionally substituted with one or more (C₁-C₄)alkyl; A² is arylene orheteroarylene, wherein A² is optionally substituted with halo; X¹ is—O—, —NH— or —N((C₁-C₄)alkyl)-; R^(1a) and R^(1b) are independently H,(C₁-C₄)alkyl, (C₂-C₄)alkenyl or (C₂-C₄)alkynyl; R² is H, (C₁-C₄)alkyl,(C₂-C₄)alkenyl or (C₂-C₄)alkynyl; R^(3a) and R^(3b) are independently Hor (C₁-C₈)alkyl; R^(4a) and R^(4b) are independently H, —OH,(C₁-C₄)alkoxy, halo(C₁-C₄)alkoxy or (C₁-C₈)alkyl; R⁵ is H, (C₁-C₄)alkyl,(C₂-C₄)alkenyl or (C₂-C₄)alkynyl, or R⁵ forms a cyclic moiety along with—N((C₁-C₄)alkyl)- of X¹ or arylene of A²; and R⁶ is H or (C₁-C₄)alkyl.2. The compound of claim 1, wherein A¹ is ethenylene, propenylene,butenylene, ethylene, propylene, butylene, oxypropylene, oxypropenylene,pyrazolylene, phenylene, pyridylene or pyrimidinylene.
 3. The compoundof claim 1, wherein A² is isoquinolinylene, phenylene or halophenylene.4. The compound of claim 1, wherein X¹ is —O— or —NH—; one of R^(1a) andR^(1b) is H and the other is methyl; R² is iso-propyl; R⁵ is methyl andR⁶ is H or methyl.
 5. The compound of claim 1, wherein R^(3a) is H ormethyl; R^(3b) is H; R^(4a) is H, —OH, methoxy, trifluoroethoxy; andR^(4b) is H.
 6. The compound of claim 1, which is a compound of FormulaII:

or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture ofstereoisomers, tautomer, ester or prodrug thereof, wherein: A¹ isethenylene,

A¹ is

X¹ is —O— or —NH—; R^(3a) is H or methyl; R^(4a) is H, —OH,(C₁-C₄)alkoxy, halo(C₁-C₄)alkoxy or (C₁-C₈)alkyl; and R⁵ is H or(C₁-C₄)alkyl.
 7. The compound of claim 1, wherein A² is heteroarylene;A¹ is (C₂-C₅)alkylene, (C₂-C₅)alkenylene, (C₂-C₅)alkynylene, wherein A¹is optionally substituted with one or more (C₁-C₄)alkyl; R^(3a) is H ormethyl; and R^(4a) is H, —OH or (C₁-C₄)alkoxy.
 8. (canceled)
 9. Thecompound of claim 1, wherein A² is arylene; and A¹ is (C₂-C₅)alkylene,(C₂-C₅)alkenylene, (C₂-C₅)alkynylene, —O—(C₂-C₅)alkylene,—O—(C₂-C₄)alkenylene, wherein A¹ is optionally substituted with one ormore (C₁-C₄)alkyl; R^(3a) is H or methyl; and R^(4a) is H, —OH,(C₁-C₄)alkoxy or halo(C₁-C₄)alkoxy.
 10. A compound selected from:

or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture ofstereoisomers, tautomer, ester or prodrug thereof.
 11. The compound ofclaim 1, wherein A² is arylene; and A¹ is pyrazolylene, phenylene orpyridylene.
 12. (canceled)
 13. The compound of claim 1, wherein A² ishalophenylene; and A¹ is —O—(C₂-C₅)alkylene or —O—(C₂-C₄)alkenylene. 14.(canceled)
 15. The compound of claim 1, wherein A¹ is (C₂-C₅)alkylene or(C₂-C₄)alkenylene; R⁵ is methyl, or R⁵ form

along with arylene of A², or R⁵ form

along with —N((C₁-C₄)alkyl)- of X¹; and R⁶ is H or methyl.
 16. Acompound selected from:

or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture ofstereoisomers, tautomer, ester or prodrug thereof.
 17. A pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof claim 1 or a pharmaceutically acceptable salt, isotope, stereoisomer,mixture of stereoisomers, tautomer, ester or prodrug thereof and apharmaceutically acceptable excipient.
 18. The pharmaceuticalcomposition of claim 17, further comprising at least one additionaltherapeutic agent selected from the group consisting of interferons,ribavirin, HCV NS3 protease inhibitors, HCV NS5a inhibitors, nucleosideor nucleotide inhibitors of HCV NS5B polymerase, non-nucleosideinhibitors of HCV NS5B polymerase, and TLR-7 agonists; or a mixturethereof.
 19. The pharmaceutical composition of claim 18, wherein the atleast one addition therapeutic agent is ribavirin, telaprevir,boceprevir or sofosbuvir. 20.-26. (canceled)